# 


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i . Books  aro  not  to  be  taken  from  tlie  Library. 

^ # 

Accession  No. a 


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Digitized  by  the  Internet  Archive 
in  2016 

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OUTLIITES 


OF 

PRACTICAL  HISTOLOGY. 


MORRIS’  ANATOMY. 

791  Illustrations,  Many  in  Colors. 

A Complete  Text-Book.  Edited  by  Heney  Moeris,  f.  e.  c.  s.  , Surgeon 
to,  and  Lecturer  on  Anatomy  at,  Middlesex  Hospital,  assisted  by  J.  Bland 
Sutton,  f.r.c.s.,  J.  H.  Davies-Colley,  f.e.c.s.,  Wm.  J.  Walshman, 
F.R.C.S.,  H.  St.  John  Brooks,  m.d.,  R.  Marcus  Gunn,  f.e.c.s.,  Arthur 
Hensman,  f.r.c.s.,  Frederick  Treves,  f.e.c.s.,  William  Ander- 
son, F.E.C.S.,  and  Prof.  W.  H.  A.  Jacobson.  One  Handsome  Octavo 
Volume,  with  971  Illustrations,  214  of  which  are  printed  in  colors. 

Cloth,  net^  $6.00;  Leather,  net^  $7.00 

***  This  Treatise  is  designed  fof^the  use  of  students,  and  is  a sys- 
tematic and  complete  description  of  all  parts  of  the  human  body,  and 
includes  a thorough  Section  by  Jacobson  on  Surgical  and  Topographical 
Anatomy,  with  130  illustrations.  Whilst  each  author  is  alone  responsible 
for  the  subject-matter  of  the  article  which  follows  his  name,  all  have 
combined,  by  reading  proofs,  by  consultation,  etc.,  to  give  uniformity  to 
the  work. 

In  no  medical  text-book  of  late  date  is  there  such  a wealth  of  illus- 
tration. Recognizing  at  the  outset  that  this  was  a most  important  part  of 
the  work  in  hand,  no  expense  was  spared  in  its  development.  With  very 
few  exceptions,  all  the  illustrations  have  been  drawn  and  engraved  specially 
for  this  purpose  from  original  dissections.  Colors  have  been  freely  used 
in  the  printing  in  order  to  bring  out  all  important  details. 

“The  illustrations  are  profuse  and  well  executed,  numbering  in  all  seven  hundred 
and  ninety-one  wood-cuts,  two  hundred  and  fourteen  of  which  are  in  colors  from  origi- 
nal sketches.  While  it  must  be  admitted  that  there  can  be  scarcely  anything  new  in 
descriptive  anatomy,  it  is  refreshing  to  notice  a departure  in  the  arrangement  and  treat- 
ment of  this  subject,  which  is  practical,  useful,  and  interesting.  In  a word,  the 
natural  method  is  adopted,  and  several  new  features  of  illustration  are  introduced.  For 
instance,  the  origin  and  insertion  of  muscles  with  exact  areas  of  attachment  are  noted  in 
different  colored  outlines,  red  for  the  former  and  blue  for  the  latter.  Thus  the  reader 
is  enabled  at  a glance  to  discriminate  in  the  case  of  a given  bone  the  difference  between 
the  two  points,  and  obtain  thereby  the  direction  and  extent  of  action  of  each  muscle.  A 
similar  principle  is  carried  out  in  other  parts  of  the  work,  which  makes  the  illustrations 
for  the  most  part  unique  and  invaluable,  the  schematic  drawings  especially  demanding 
attention  and  commendation  in  this  connection.  The  work  as  a whole  is  filled  with  prac- 
tical ideas,  and  the  salient  points  of  the  subjects  are  properly  emphasized.  The  surgeon 
will  be  particularly  edified  by  the  section  on  the  topographical  anatomy,  which  is  full  to 
repletion  of  excellent  and  useful  illustrations.” — New  York  Medical  Record. 

41®^  Descriptive  Circular  with  sample  pages 
and  illustrations  free  upon  application. 

P.  BLAKISTON,  son  & COm  Publishers,  Philadelphia. 


OUTLINES 


OF 

PRACTICAL  HISTOLOGY 

a fiDanual  for  StuOento, 


BY 


WILLIAM  STIRLING,  M.D.,  Sc.D., 

BRACKENBURY  PROFESSOR  OF  PHYSIOLOGY  AND  HISTOLOGY  IN  THE  OWENS  COLLEGE 
AND  PROFESSOR  IN  THE  VICTORIA  UNIVERSITY,  MANCHESTER  ; EXAMINER  IN 
PHYSIOLOGY  IN  THE  UNIVERSITIES  OF  OXFORD  AND  EDINBURGH, 

FOR  THE  FELLOWSHIP  OF  THE  ROYAL  COLLEGE  OF  SURGEONS,  ENGLAND. 


SelitI)  368  Illustration)*. 


SECOND  EDITION,  REVISED  AND  ENLARGED 


PHILADELPHIA: 

P.  BLAKISTON,  SON  & CO., 

1012  WALNUT  STREET. 

1897. 


(oH  . 

5iSo 

PREFATORY  NOTE  TO  THE  SECOND  EDITION. 


In  the  Second  Edition  of  this  Handbook  I have  endeavoured  to 
introduce  a succinct  account  of  the  more  recent  Histological 
Methods.  Most  of  these  I have  tested,  a matter  of  no  incon- 
siderable labour.  The  fruitful  methods  of  Golgi,  Ram6n  y Cayal, 
and  Ehrlich  are  fully  set  forth  in  the  text. 

I have  availed  myself  freely  of  the  Journals  and  Text-books 
published  at  home  and  abroad,  and  more  especially  useful  to  me 
have  been  the  works  of  Garbini  and  Ramon  y Cayal.  For  most  of 
the  additional  illustrations  I am  indebted  to  the  Geicehelelire  of 
Schiefferdecker  and  Kossel. 

In  conclusion,  I have  to  thank  my  Senior  Demonstrator,  James 
A.  Menzies,  M.B.,  C.M.,  for  assistance  in  reading  the  proof-sheets. 

WILLIAM  STIRLIiS^G. 


Physiological  Laboratory,  The  Owens  College, 
Manchester, 


PREFACE. 


The  present  work  was  written  primarily  to  meet  the  wants  of 
the  students  attending  the  course  of  instruction  in  “ Practical 
Histology  in  The  Owens  College,  but  it  is  hoped  that  it  will 
be  found  useful  also  to  students  of  medicine  and  science  in  other 
colleges  and  universities. 

The  Exercises  printed  in  small  type  are  intended  for  Senior 
Students,  and  for  those  attending  the  course  of  “ Advanced 
Histology  given  in  The  Owens  College. 

Although  a drawing  accompanies  almost  every  exercise,  still 
this  is  not  intended  to  relieve  the  student  from  what  is  a most 
important  part  of  the  training  in  Practical  Histology,  viz.,  that 
the  student  should  make  sketches  of  his  preparations.  It  serves 
very  little  useful  purpose  to  give  students  sections  ready  pre- 
pared, and  ask  them  merely  to  mount  them.  Hence,  consider- 
able stress  has  been  laid  on  methods^  as  a knowledge  of  these  is, 
after  all,  one  of  the  most  important  parts  of  a practical  training 
in  Histology.  Many  methods  have  been  tried  and  found  wanting, 
and  accordingly  those  only  are  introduced  which  the  author,  after 
experiment,  has  found  to  be  reliable. 

The  author  is  indebted  to  the  various  Manuals  of  Histology 
published  in  this  country,  and  also  to  those  of  Friedlander  and 
Eberth,  Stohr,  Fol,  Kolliker,  Martinotti,  Franco tte,  Bolles  Lee 
and  Henneguy,  Orth,  and  Edinger,  as  well  as  the  various  Micro- 
scopical Journals,  British  and  Foreign. 

Most  of  the  illustrations  have  been  drawn  from  microscopical 
preparations  made  in  the  class  of  Practical  Histology,  but  a few 


Vlll 


PREFACE. 


of  them  are  taken  from  preparations  kindly  presented  to  me  by 
my  friend  Professor  Swaen  of  Liege.  I am  greatly  indebted  to 
my  demonstrator,  Arthur  Clarkson,  M.B.,  C.M.,  for  a considerable 
number  of  the  drawings,  and  also  to  my  pupil,  Mr.  C.  E.  M.  Lowe, 
for  similar  services.  A few  of  the  drawings  were  made  by  myself. 
To  F.  W.  Stansfield,  M.B.,  Ch.B.  (Yict.),  my  thanks  are  due  for 
assistance  in  reading  the  proof-sheets. 

I have  also  to  express  my  obligations  to  several  scientific  in- 
strument makers  and  others,  including  Messrs.  Zeiss,  Hartnack, 
Yerick,  Leitz,  Beichert,  Jung,  Hicks,  Eternod,  Zimmerniann, 
Hume,  Beck,  Swift  & Son,  Gardner,  and  A.  Fraser. 

Finally,  I have  to  thank  my  publishers  for  the  liberal  manner 
in  which  the  work  is  illustrated. 

WILLIAM  STIBLESTG. 


Physiological  Laboratory,  The  Owens  College, 
Manchester, 


V 


COWTEI^TS, 


PART  I. 

CHAP.  PAGE 

I.  APPARATUS  EEQUIRED  . I 

IT.  THE  MICROSCOPE  AND  ITS  ACCESSORIES 5 

III.  NORMAL  OR  INDIFFERENT  FLUIDS  . ...  . .24 

IV.  DISSOCIATING  FLUIDS 24 

V.  HOW  TO  TEASE  A TISSUE 26 

VI.  FIXING  AND  HARDENING  FLUIDS 27 

VII.  DECALCIFYING  FLUIDS 36 

VIII.  PREPARING  TISSUES  FOR  MICROSCOPICAL  EXAMINATION  . . 38 

IX.  EMBEDDING  . . , 40 

X.  SECTION  CUTTING 48 

XI.  FIXATIVES  ...  60 

XII.  STAINING  REAGENTS  . 63 

XIII.  CLARIFYING  REAGENTS  82 

XIV.  MOUNTING  FLUIDS  AND  METHODS 85 

XV.  INJECTING  BLOOD-VESSELS  AND  GLAND-TUBES  ....  89 

XVI.  EXAMINATION  OF  FRESH  TISSUES  AND  FLUIDS  ....  92 


PAET  II. 

LESSON 

I.  MILK*  GRANULES,  FIBRES,  AND  VEGETABLE  ORGANISMS  . . 97 

II.  THE  BLOOD I06 

III.  HUMAN  BLOOD,  CRYSTALS  FROM  BLOOD,  AND  BLOOD  PLATELETS  II5 

IV.  EPITHELIUM  (STRATIFIED)  AND  ENDOTHELIUM  . . . . I24 

V.  COLUMNAR,  SECRETORY,  AND  TRANSITIONAL  EPITHELIUM  . . I31 

VI.  CILIATED  EPITHELIUM 1 35 

VII.  KARYOKINESIS  OR  MITOSIS I4I 

VIII.  CELLULAR  AND  HYALINE  CARTILAGE I46 

IX.  THE  FIBRO-CARTILAGES,  WHITE  AND  YELLOW  . , . . I5I 

X.  CONNECTIVE  TISSUE 1 56 

XI.  TENDON 163 


X 


CONTENTS. 


LESSON  i*AGE 

XII.  ADIPOSE,  MUCOUS,  AND  ADENOID  TISSUES— PIGMENT-CELLS  . l68 

XIII.  BONE,  OSSEOUS  TISSUE . 174 

XIV.  BONE  AND  ITS  DEVELOPMENT  ......  182 

XV.  MUSCULAR  TISSUE 1 89 

XVI.  STRIPED  OR  STRIATED  MUSCLE  . , , . » • 193 

XVII.  NERVE-FIBRES 202 

XVIII.  NERVE-CELLS,  NERVE-GANGLIA,  AND  PERIPHERAL  TERMINA- 
TIONS OF  MOTOR  NERVES 213 

XIX.  THE  HEART  AND  BLOOD-VESSELS 223 

XX.  THE  LYMPHATIC  SYSTEM,  SPLEEN,  AND  THYMUS  GLAND  . 234 

XXI.  TONGUE,  TASTE  BUDS,  SOFT  PALATE  .....  246 

XXII.  TOOTH,  (ESOPHAGUS . - 25 1 

XXIII.  SALIVARY  GLANDS  AND  PANCREAS 256 

XXIV.  THE  STOMACH 266 

XXV.  THE  SMALL  AND  LARGE  INTESTINE  , . , . - 272 

XXVI.  LIVER 285 

XXVII.  TRACHEA,  LUNGS,  THYROID  GLAND 294 

XXVIII.  KIDNEY,  URETER,  BLADDER,  SUPRARENAL  CAPSULE  . , 303 

XXIX.  SKIN  AND  EPIDERMAL  APPENDAGES 315 

XXX.  SPINAL  CORD 328 

XXXI.  MEDULLA  OBLONGATA,  CEREBELLUM,  CEREBRUM  . . , 347 

XXXII.  THE  EYE  . 358 

XXXIII.  EAR  AND  NOSE 37 1 

XXXIV.  TERMINATION  OF  NERVES  IN  SKIN  AND  MUCOUS  MEMBRANES  376 

XXXV.  THE  TESTIS 381 

XXXVI.  OVARY,  FALLOPIAN  TUBE,  UTERUS 388 

XXXVII.  MAMMARY  GLAND,  UMBILICAL  CORD,  AND  PLACENTA  . . 393 

XXXVIII.  TO  MAKE  PREPARATIONS  RAPIDLY  FROM  FRESH  TISSUES  . 396 


APPE^^DIX. 

A.  — Some  General  Works  of  Reference 406 

B.  — Tables  of  Magnifying  Power  of  Objectives  and  Oculars  . 408 

C.  —List  of  Makers,  British  and  Foreign,  of  Microscopes, 

Microtomes,  Chemicals,  and  Histological  Reagents  . 409 

D.  — Weights  and  Measures,  Table  of  Equivalents  . . .410 

Index . 411 


LIST  OF  ILLUSTRATIONS. 


VIG.  PAGE 

1.  English  form  of  slide.  {Z-eiss.)  . . . . . . . i 

2.  S([uare  and  round  cover-glasses.  (Zeiss.)  .....  i 

3.  Pinewood  cabinet.  (Swift. ) . . • . . • . .2 

4.  Paper  tray  for  slides.  (Jung.)  .......  2 

5.  Scissors.  {Beck.)  . . . . ‘ . . . . .3 

6.  Cover-glass  lifter  .........  3 

7.  Cornet’s  cover-glass  forceps  .......  3 

8.  Section-lifter.  (Beck.)  ........  4 

<j.  Leitz’s  microscope,  No.  5.  (Leitz  ) . . . . . • ^ m 

10.  Zeiss’s  large  stand.  (Zeiss.)  .......  8 

11.  Iris,  diapliragni.  (Zeiss.)  .......  9 

12.  Diaphragms.  (Reichert.)  ........  9 

13.  Abbe’s  condenser.  (Sicijft.)  . . . . . . .11 

14.  J eitz’s  Ia  stand.  (Leitz.)  .......  12 

15.  Swift’s  college  microscope.  (Swift.)  ......  14 

16.  Hartnack’s  No.  III.  ........  15 

17.  Zeiss’s  camera  lucida.  (Zeiss.)  .......  17 

18.  Abbe’s  camera  lucida.  (Zeiss.)  . . . . _ . . .17 

19.  Chevalier’s  camera  lucida.  (Verick.)  ......  18 

20.  Malassez’s  camera.  {Verick.)  .......  19 

21.  Eye-piece  micrometer  scale.  (Zeiss.)  ......  20 

22.  Eye-piece  micrometer.  (Zeiss.)  . . . . . . .21 

23.  Microscope  lamp.  (Swift.)  .......  21 

24.  Dissecting  microscope.  (Verick.)  ......  22 

25.  Eeichert's  dissecting  microscope  .......  22 

26.  Cover-glass  tester.  (Zeiss.)  . . . . . . • ^3 

27.  Glass-thimble.  (Beck.)  ....  ...,  26 

28.  Photophore.  (Stirling.)  ........  27 

29.  IMayer’s  embedding  b ith.  (Jung.)  ......  42 

30.  Embedding  hox.  (Stirling.)  .......  45 

31.  Celloidin  embe  Iding  box.  (Stirling.)  ......  46 

32.  Valentine’s  knife.  (Beck.)  .......  49 

33.  Kutherford's  freezing  microtome.  (Gardner.)  . . . .50 

34.  Catbcart’s  freezing  microtome.  (Fraser.)  .....  52 

35.  Planing-iron.  (Reichert.)  .......  53 

36.  Malassez’s  microtome.  (Verick.)  ......  54 

37.  Cambridge  rocking  microtome  .......  54 

38.  Minot’s  microtome.  (Zimmermann.)  ......  55 

39.  Silk  band  for  serial  sections.  (Zimmermann.)  . . . . 5^ 

40.  Thoma’s  sledge  microtome.  (Jung.)  ......  56 

41.  Malassez’ microtome.  (Verick.)  ......  57 

42.  Williams’  microtome.  (Swift.)  .......  57 

43.  Swift’s  ether  microtome.  (Swift.)  ......  58 

44.  Kanvier’s  hand-microtome.  (Reichert.)  .....  59 

45.  Vulcanite  rings.  (Eternod.)  .......  77 

46.  Ring  for  stretching  membranes.  (Eternod.)  .....  77 

47.  Pvanvier’s  support.  (Ranvier.)  .......  82 

48.  Capped  balsam  bottle.  (Beck.)  .......  86 

49.  Turntable.  (Beck.)  ........  88 

50.  Turntable  with  slide.  ........  88 

51.  Hand-centrifuge.  (Muencke.)  .......  94 

52.  Milk  globules.  .........  99 

53.  Potato  starch.  (Stirling.)  .......  100 

54.  Rice  starch.  (Blyth.)  . . . . . , . .101 

55.  Air,  fat  granules  in  water.  (Ranvier.)  . . . , .101- 


Xll 


LIST  OF  ILLUSTRATIONS. 


FIG. 

56.  Silk,  wool,  cotton,  linen.  {Stirling.) 

57.  Cells  from  onion-bulb.  {Schieffer decker.) 

58.  Mould.  {Ainsworth  Davis.) 

59.  Yeast-cells.  {Landois  and  Stirling.)  . 

60.  Micrococci.  ( V.  Jaksch. ) 

61.  Blood  of  frog.  {Ranvier.) 

62.  Amphibian  blood-corpuscles.  (Landois  and  Stirling. ) 

63.  Action  of  acetic  acid  on  blood.  (Stirling.)  . 

64.  Action  of  water  on  blood-corpuscles.  (Stirling.) 

65.  Action  of  syrup  on  frog’s  blood.  (Stirling.)  . 

66.  Tannic  acid  on  blood.  (Stirling.) 

67.  Boracic  acid  on  blood.  (Stirling.) 

68.  Blood-corpuscles  of  lish  and  bird.  (Stirling.) 

69.  Amoeboid  movements  of  leucocytes,  (Landois.) 

70.  Moist  chamber.  (Ranvier.) 

71.  Acetic  acid  on  colourless  corpuscles.  (Stirling.) 

72.  Human  blood.  ( Ranvier. ) . . . 

73.  Human  blood.  (Landois.) 

74.  Human  red  corpuscles.  (Ranvier.) 

75.  Copper  hot  stage.  (Stirling.)  . 

76.  Reichert’s  warm  stage.  (Reichert.) 

77.  Crenation  of  corpuscles.  (Stirling.)  . 

78.  Fibrils  of  fibrin.  (Landois.) 

79.  Rat’s  hsemoglobin.  (Stirling.)  . 

80.  Guinea-pig’s  luemoglobin.  {Landois  and  Stirling. 

81.  Hiemin  crystals.  (V.  Jaksch.)  , 

82.  Leukaemic  blood.  (V.  Jaksch.) 

83.  Blood  platelets.*  (Landois  and  Stirling.) 

84.  Isolated  epithelial  squames 

85.  Squames  of  newt’s  epidermis.  (Stirling.) 

86.  Hard  palate  of  cat.  (Stirling.) 

87.  Prickle-cells  of  epidermis.  (Stirling.) 

88.  Isolated  prickle-cells.  (Ranvier.) 

89.  Endothelium  of  central  tendon.  (Ranvier.) 

90.  Omentum  of  young  rabbit,  silver  nitrate.  (Stirling. ) 

91.  Omentum  of  cat,  silvered.  (Stirling.) 

92.  Columnar  and  goblet  cells.  (Stirling.) 

93.  Isolated  columnar  cells.  (Stirling.) 

94.  Liver-cells.  (Cadiat.)  . 

95.  Transitional  cells.  (Stirling.)  . 

96.  Gas  chamber.  (Gscheidlen.) 

97.  Gas  chamber.  (Gscheidlen.) 

98.  Ranvier’s  moist  chamber.  ( Verick. ) 

99.  Ciliated  epithelium.  (Stirling.) 

100.  Frog’s  ciliated  cell.  (Ranvier.) 

101.  Ciliated  epithelium,  ox.  (Stirling.) 

102.  Y.S.  ciliated  epithelium 

103.  Goblet-cells.  (Stirling.) 

104.  V. S.  frog’s  tongue.  (Stirling.) 

105.  Connective-tissue  corpuscle.  (Stohr.) 

106.  Mitosis  .... 

107.  V.S.  epidermis  of  salamander.  (Stirling.) 

108.  Mitosis.  (Stirling.) 

109.  Cellular  cartilage.  (Stirling.)  . 
no.  Hyaline  cartilage.  (Stohr.) 

111.  Hyaline  cartilage  of  thyroid.  (Schieffer decker.) 

1 1 2.  Costal  cartilage.  (Stohr.) 

113.  V.S.  articular  cartilage.  (Stirling.) 

1 14.  Branched  cartilage-cells.  (Stirling.) 

115.  V.S.  intervertebral  disc.  (Stirling.) 

1 16.  White  fibro-cartilage.  (Stohr.) 

117.  T.S.  epiglottis.  (Ranvier.) 
ti8.  Elastic  cartilage  ear  of  horse.  (Schieffer decker.) 

1 19.  Transition  from  hyaline  to  elastic  cartilage.  (Schieffer decker.) 


PAGE 

102 

103 

104 
104 
104 
107 

107 

108 
108 

108 

109 
109 
no 
in 
in 
112 
116 
116 
116 
118 

118 

119 

120 
120 

120 

121 
121 
123 

125 

126 

127 

128 

128 

129 

129 

130 

132 

133 

133 

134 

136 

137 
137 


138 

139 

139 

140 

142 

143 

144 
144 

146 

147 

148 

149 

150 

151 

152 

153 

154 

155 
155 


LIST  OF  ILLUSTRATIONS.  xiii 

FIG.  PAGE 

120.  Elastic  fibres.  {Stohr.)  . . . . . . . .157 

121.  T.S.  ligamentiim  niichie.  {Stirling.)  ......  158 

122.  Elastic  fibres.  {Stirling.)  .......  158 

123.  Fenestrated  membrane.  {Stohr.)  ......  159 

124.  Areolar  tissue.  {Schiefferdecker.)  ......  159 

125.  Omentum  of  dog.  {SMeffer decker,)  . . . . . . 160 

126.  Hypodermic  syringe.  {Hicks.)  ......  i6r 

127.  Coarsely  granular  cells.  {Stirling.)  ......  162 

128.  Cell-spaces  in  areolar  tissue.  iStirling.)  .....  162 

129.  T.S.  tendon.  {Stirling.)  .......  163 

130.  L.S.  tendon.  {Schiefferdecker.)  ......  164 

131.  Fibrils  of  tendon.  {Stirling.)  .......  165 

132.  Tendon  of  rat’s  tail.  {Stirling.)  ......  165 

133.  Tendon  cells,  {lianvier.)  . . • . . . . . 165 

134.  T.S.  tendon  of  rat’s  tail.  {Stirling.)  ......  166 

135.  Endothelial  slieatb  of  tendon.  {Stirling.)  .....  166 

136.  Cell-spaces  of  diaphragm.  {Stirling.)  .....  167 

137.  Fat-cells.  {Stirling.)  ........  169 

138.  Fat-cells.  {Schiefferdecker.)  .......  169 

139.  Empty  fat-cells.  {Stirling.)  .......  170 

140.  Fat-cells  with  margarine  crystals.  ......  170 

141.  Development  of  fat-cells.  {Stirling.)  . .....  171 

142.  Mucous  tissue.  {Stirling. ) ......  . 172 

143.  Adenoid  tissue.  {Stohr.)  .......  173 

144.  Pigment  and  guanin  cells.  {Stirling.)  .....  173 

145.  T.S.  metacarpal  bone.  (Ranvier.)  ......  175 

146.  T.S.  femur  {Ranvier.)  ........  176 

147.  T.S.  dense  bone.  {Ranvier.)  .......  'l'jj 

148.  L.S.  dense  bone.  {Stirling.)  .......  177 

149.  T.S.  metacarpal  bone.  {Stohr.)  ......  178 

150.  T.S.  Haversian  canal.  {Schiefferdecker.)  .....  179 

151.  Sharpey’s  fibres.  {Stirling.)  .......  179 

152.  L.S.  injected  bone.  {Stirling.)  . ......  180 

153.  Cancellated  bone.  {Stirling.)  .......  180 

154.  Cancellated  bone.  {Stirling.)  .......  181 

155.  T.S.  embryonic  bone.  {Ranvier.)  ......  182 

156.  T.S.  foetal  bone.  {Stohr.)  . . . . . . .183 

157.  L.S.  developing  bone.  {Stohr.)  ......  184 

158.  V.S.  tibia.  {Stirling.)  ........  185 

159.  Developing  bone.  {Ranvier.)  .......  186 

160.  Membranous  bone.  {Stohr.)  .......  186 

161.  Marrow  cell.  {Stirling.)  .......  187 

162.  Isolated  smooth  muscle.  {Lando is  and  Stirling.)  ....  190 

163.  Bladder  of  frog.  {Stirling.)  .......  190 

164.  T.S.  smooth  muscle.  {Stirling.)  ......  191 

165.  Cement  of  smooth  muscle.  {Stirling.)  .....  191 

166.  T.S.  smooth  muscle.  {Schiefferdecker.)  .....  192 

167.  Striped  muscle  of  frog.  {Stirling.)  ......  194 

168.  Muscle  of  fibrils.  {Schiefferdecker.)  ......  195 

169.  Tendon  and  muscle.  {Landois  and  Stirling.)  ....  196 

T70.  Muscular  fibre.  {Ranvier.)  .......  196 

171.  Fibril  of  hydrophilus.  {Ranvier.)  ......  196 

172.  Crab’s  muscle.  {Stirling.)  . ...  . . . . 197 

173.  T.S.  muscle.  {Krause.)  ........  197 

174.  T.S.  muscle.  {Stirling.)  .......  197 

175.  Injected  muscle.  {Landois  and  Stirling.)  .....  197 

176.  T.S.  and  L.S.  injected  muscle.  {Owsjannikow.)  ....  198 

177.  T.S.  injected  muscle.  {Stirling.)  . . . . . . 199 

178.  Heart  muscle.  . . . . . , . . . 199 

179.  T.S.  frozen  muscle.  ........  199 

180.  Polariser.  {Zeiss.)  ........  201 

1 8 1.  Medullated  nerve-fibre.  {Stirling.)  ......  203 

182.  Non-medullated  nerve-fibre.  {Ranvier.)  .....  204 

183.  Fresh  nerve-libre.  {Obersteiner.)  ......  205 


xiv  LIST  OF  ILLUSTRATIONS. 

J'w.  PAGE 

184.  Nerve-fibre  after  osmic  acid.  {Schivalbe.)  .....  205 

185.  Frog’s  nerve-fibre.  {Obersteiner.)  ......  206 

186.  Ranvier’s  cros;es.  [Obersteiner.)  ......  207 

187.  Ranvier’s  cross  and  From mann’s  lines.  [Obersteiner.)  . . . 207 

188.  Perijiheral  nerve-fibre  with  axis-cylinder  .....  208 

189.  Intercostal  nerve  with  Ranvier’s  crosses  .....  208 

190.  T.S.  nerve.  [Eichhorst.)  .......  209 

191.  Non-niedullated  nerve.  [Obersteiner.)  . . . . . 209 

192.  Sympathetic  nerve.  [Schieffer decker.)  .....  210 

193.  Neuro-keratin  network.  . . . . . . . .210 

194.  Frommann’s  lines.  [Obersteiner.)  ......  211 

195.  Nerve-fibre  of  frog.  [Obersteiner.)  ......  211 

196.  T.S.  nerve-fibres,  osmic  acid.  [Stirling.)  .....  212 

197.  Spinal  ganglion.  [Cadiat.)  .......  214 

198.  Cells  of  spinal  ganglion.  [Obersteiner.)  .....  214 

199.  Bipolar  nerve-cell  of  skate.  .......  215 

200.  Spinal  ganglion  nerve-cell.  [Ranvier.)  .....  216 

201.  Cervical  sympatbetic  ganglionic  cell.  [Stirling.)  ....  217 

2C2.  Multipolar  nerve-cell  of  spinal  cord.  [Obersteiner.)  . . . 218 

203.  Nerves  of  frog’s  s irtorius.  [Mays.)  ......  220 

204.  End-plates  of  lizard.  [Kuhne.)  . ....  221 

205.  End-plate  of  lizard.  [Stirling.)  ......  221 

206.  Sympathetic  nerve-cell.  [Ranvier. ) . . . . . . 222 

207.  Heart  muscle.  [Stirling.)  .......  224 

208.  Purkinje’s  fibres.  [Ranvier.)  .......  225 

209.  Endocardium.  [Ranvier.)  .......  226 

210.  T.S.  tricuspid  valve.  [Ranvier.)  ......  226 

211.  L. S.  human  aorta.  [Ranvier.)  . . . . . . 227 

212.  T.S.  artery.  [Stirling.)  .......  227 

213.  Endothelium  of  artery  and  vein.  [Stirling.)  ....  228 

214.  Capillaries.  [Obersteiner.)  . . ' . . . . . 229 

215.  Small  artery.  [Stirling.)  ..  ^ ...  . 229 

216.  Arteriole.  [Obersteiner.)  .......  229 

217.  T.S.  small  artery  and  vein.  [Stirling.)  .....  230 

218.  Artery  of  brain.  [Obersteiner.)  ......  231 

219.  Arteriole,  silvered.  [Ranvier.)  ......  231 

220.  Capillary,  silvered.  [Landois  and  Stirling .)  ....  232 

221.  Developing  blood- vessels.  [Stirling.)  .....  233 

222.  L. S.  lymph  gland.  [Ranvier.)  ......  235 

223.  Lymph  sinuses.  [Stirling.)  .......  237 

224.  Central  tendon.  [Ranvier.)  .......  238 

225.  Pleural  surface  of  diaphragm.  [Stirling.)  .....  238 

226.  Stomata.  [Stirling.)  ........  239 

227.  Tonsil.  [Stohr.)  .........  240 

228.  Thymus.  [Stirling.)  ........  241 

229.  Injected  thymus.  [Cadiat.)  .......  242 

230.  Elements  of  thymus.  [Cadiat.)  ......  242 

231.  Human  spleen.  [Stohr.)  . . - . . . . . 243 

232.  Elements  of  splenic  pulp.  [Stohr.)  ......  244 

233.  Reticulum  of  spleen.  [Cadiat.)  ......  244 

234.  T.S.  tongue  of  cat.  (Stirling.)  ......  246 

235.  Filiform  papilla.  [Stohr.)  .......  247 

236.  Fungiform  papilla.  [Stohr.)  *......  247 

237.  Crypt  of  tongue.  [Schenk  ) ......  . 248 

238.  Injected  tongue  of  cat.  [Stirling.)  ......  248 

239.  Papillae  foliatse.  [Stirling.)  .......  249 

240.  V.S.  Papillae  foliatae.  [Stohr.)  ......  250 

241.  V.S.  tooth  . . . . . . . . . . 252 

242.  243.  Enamel  prisms.  [Landois  and  Stirling)  . . . . 253 

244.  T.S.  fang  of  tooth.  [Landois  and  Stirling.)  . . . . 253 

245.  Development  of  tooth.  [Landois  and  Stirling.)  ....  254 

246.  Later  stage  of  245.  [Stbhr.)  .......  254 

247.  Later  stage  of  246.  [Stohr.)  .......  254 

248.  T.S.  oesophagus.  [Stirling.)  .......  255 


LIST  OF  ILLUSTRATIONS. 


XV 


FIG. 

249.  Lobules  of  submaxillary  gland.  {Stirling.)  . 

250.  A^'ini  of  submaxillary  gland.  {Stirling.) 

251.  T.S.  duct  of  salivaiy  gland.  {Landois  and  Stirling.) 

252.  Resting  serous  glaiii  {Heidenhain.) 

253.  Human  submaxillary  gland.  {Heidenhain.) 

254.  T.S.  pancreas.  {Stirling.)  .... 

255.  T.S.  fresh  pancreas.  {Heidenhain.)  . 

256.  V.S.  stomach.  {Stohr.)  ..... 

257.  V.S.  mucous  membrane  of  stomach.  {Stirling.) 

258.  T.S.  gastric  glands.  {Landois  and  Stirling.) 

259.  V.S.  pyloric  glands.  [Heidenhain.)  . 

260.  T.S.  small  intestine.  {Stirling.) 

261.  L.S.  Peyer’s  patch.  {Landois  and  Stirling.) 

262.  Injected  small  intestine.  {Stohr.) 

263.  Scheme  of  262.  {Mall.).  . . 

264.  Injected  villi.  [Stirling.)  .... 

265.  Auerbach’s  plexus.  [Stirling.)  . 

266.  Auerbach’s  plexus.  [Cad  at.)  .... 

267.  Section  of  266.  {Cadiat.)  .... 

268.  Meissner’s  plexus.  {Stirling.)  .... 

269.  V.S.  duodenum.  [Stohr.)  .... 

270.  L.S.  large  intestine.  [Schenk.). 

271.  Lieberklilm’s  gland.  [Heidenhain.)  . 

272.  T.S.  villus.  [Heidenhain.)  .... 

273.  Intestinal  villus.  [Kultschitzky .) 

274.  Villus  absorbing  fat.  [Stirling.) 

275.  T.S.  liver  of  pig.  [Stirling.)  .... 

276.  Liver-cells.  [Heidenhain}^  .... 

277.  T.S.  portal  canal.  [Stirling.)  .... 

278.  Human  liver.  [Stohr.)  ..... 

279.  Liver  of  frog.  {Stirling.)  .... 

280.  Injected  liver  of  rabbit.  [Stirling.)  . 

281.  Interlobular  bile-duct.  [Landois  and  Stirling.) 

282.  B le-ducts  iniected.  [Cadiat.)  .... 

283.  T.S.  trachea  of  cat.  [Stirling.) 

284.  L S.  trachea  of  cat.  [Stirling.) 

285.  T.S.  bronchus.  [Stirling.)  .... 

286.  T.S.  human  bronchus.  [Hamilton.)  . 

287.  V.S.  lung  and  pleura.  [Hamilton.)  . 

288.  Silvered  lung  of  kitten.  [Hamilton.)  . 

289.  Injected  lung.  [Stirling.)  .... 

290.  T.S.  dried  lung.  [Stirling.)  .... 

291.  T.S.  foetal  lung.  [Stirling.)  .... 

292.  T.S.  thyroid  gland.  [Cadiat.)  .... 

293.  L.S.  Malpighian  pyramid.  .... 

294.  Scheme  of  renal  tubules.  [Klein.) 

295.  Glomerulus.  [Stirling.)  .... 

296.  Rodded  epithelium.  [Landois  and  Stirling .) 

297.  Irregular  renal  tubule.  [Landois  and  Stirling.) 

298.  T.S.  apex  of  Malpighian  pyramid.  [Stirling.) 

299.  Blood-vessels  of  kidney.  [Land  is  and  Stirling. ) . 

300.  Henle’s  tubule.  ( Landois  and  Stirling. ) 

301.  Cells  from  renal  tubule.  [Landois  and  Stirling .)  . 

302.  T.S.  ureter.  [Stohr.)  ..... 

303.  V S.  bladder.  [Stohr.)  ..... 

304.  T.S.  penis.  (Stirling.)  ..... 

305.  V.S.  suprarenal  capsule.  (Stohr.) 

306.  V.S.  skill  of  palm.  [Stirling.). 

307.  Human  epidermis.  (Ranvier.  ) . 

308.  T.S.  sweat  gland.  (Stirling.)  .... 

309.  V.S.  hair-follicle.  [Landois  and  Stirling.)  . 

310.  T.S.  hair-follicle.  [Landois  and  Stirling .)  . 

31 1.  V.S.  injected  skin.  [Taguschi.) 

312.  T.S.  nail.  [Landois  and  Stirling.) 


PAGB 

260 

260 

261 

261 

262 

263 

264 

268 

269 

270 
270 

274 

275 

276 

277 

277 

278 

278 

279 

279 

280 

281 

281 

282 

283 


287 

287 

288 

289 

290 

290 

291 

295 

296 

296 

297 

298 

299 

299 

300 

300 

301 
3^3 

304 

308 

308 

308 

309 

310 

311 

311 

312 

313 

313 

314 

317 

318 

319 

322 

324 

324 

326 


XVI 


LIST  OF  ILLUSTRATIONS. 


FIG. 

313.  Axillary  gland.  {Landois  and  Stirling.) 

314.  T.S.  spinal  cord.  {Cadiat.)  .... 
315  T.S.  anterior  cornu  of  cord  {Ohersteiner .)  . 

316.  T.  S.  white  matter  of  cord.  Ohersteiner.) 

317-20.  T.S.  spinal  cord  at  various  levels.  (Ohersteiner.) 

321.  Neuroglia  cell.  (Ranvier.)  .... 

322.  L.S.  spinal  cord  to  show  collateral  fibres.  (Kolliker.) 

323.  T.S.  medulla  oblongata.  (Henle.) 

324.  T.S,  medulla  oblongata.  (Edinger.)  . 

325.  Leaflet  of  cerebellum.  (Stirling. ) . . . 

326.  V.S.  cerebellum.  (Ohersteiner.) 

'^'27,  V.S.  cerebrum.  (Ohersteiner.)  . 

328.  V.S.  frontal  convolution.  (Ohersteiner.) 

329.  V.S.  injected  cerebrum.  (Ohersteiner.) 

330.  Cell  of  Purkinje.  (Ohersteiner.) 

331.  T.S.  cerebrum  of  rat.  (Cayal.) 

332.  V.S.  cerebrum.  (Edinger.)  .... 
333-  V.S.  cornea.  (Landois  and  Stirling .) 

334.  Cornea  corpuscles.  (Ranvier.) 

335.  Nerves  of  cornea.  (Ranvier.)  . . . . 

336.  V.S.  cornea.  (Ranvier.)  .... 

337.  Cornea  cell-spaces.  (Ranvier.) 

338.  V.S.  sclerotic  and  choroid.  (Stohr.)  . 

339.  T.S.  sclerotic  and  cornea.  (Landois  and  Stirling.) 

340.  Lens  fibres.  ( Landois  and  Stirling. ) . 

341.  V.S.  retina.  (Ranvier.)  .... 

342.  V.S.  cochlea.  (Ranvier.)  .... 

343.  V.S.  cochlear  duct.  (Landois  and  Stirling .) 

344.  V.S.  olfactory  mucous  membrane.  (Stohr.)  . 

345.  Olfactory  ceils.  (Landois  and  Stirling .) 

346.  Tactile  cells.  (Ranvier.)  .... 

347.  Tactile  discs.  (Ranvier. ) ...  . 

348.  End-bulb  ....... 

349.  Pacini’s  corpuscle.  (Ranvier.) 

350.  Endothelium  of  349.  (Stirling.) 

351.  T.S.  of  349.  (Stirling.) 

352.  Wagner’s  corpuscle.  (Landois  and  Stirling.) 

353.  Wagner’s  corpuscle.  (Ranvier.) 

354.  Organ  of  Eimer.  (Stirling.)  .... 

355.  T.S.  testis  ....... 

356.  Tubule  of  testis.  (Landois  and  Stirling .) 

357.  Spermatogenesis  of  rat.  (Stirling.) 

358.  Spermatozoa.  (Landois  and  Stirling .) 

359.  Epididymis.  (Schenk.)  ..... 

360.  V.S.  ovary.  (Turner.)  ..... 

361.  Ovum  ....... 

362.  T.S.  Fallopian  tube.  (Schenk.) 

363.  T. S.  fimbriated  end  of  362.  (Stirling.) 

364.  V.S.  uterus.  (Stirling.)  .... 

365.  T.S.  mamma.  (Stirling.)  .... 

366.  T.S.  active  mamma.  (Stirling.) 

367.  Colostrum.  (V.  Jaksch.)  .... 

368.  Placental  villus.  (Cadiat.)  .... 


PAGE 

327 

334 

335 

335 

336 
343 

347 

348 
348 
350 
350 
352 
352 
354 
356 

356 

357 
359 

359 

360 

360 

361 

361 

362 

364 

366 

372 

373 
375 
375 

377 

378 

378 

379 
379 

379 

380 

381 

381 

382 
382 

386 

387 
387 

389 

390 

391 

391 

392 
394 
394 

394 

395 


[The  illustrations  indicated  by  the  word  Stohr''  are  from  Stbhr’s  Lehrhuch 
der  Histologie ; by  '•'‘Cadiat''  from  Cadiat’s  Traits  d' Airmtomie  Generate;  by 
"Ranvier''  from  Ranvier’s  Traite  Technique  d' Histologie ; by  "Schenk,"  from 
Schenk’s  Grundriss  der  normalen  Histologie  ; by  “ Ohersteiner,"  from  Obersteiner’s 
Anleitung  heim  Studium  des  Baues  dernervosen  Centralorgane  ; by  " v.  Jaksch," 
from  V.  Jaksch’s  Klinische  Diagnostik ; by  "Edinger,"  from  Edinger’s 
Vorlesungen  uher  den  Bau  der  nervosen  Centralorgane ; by  “ Landois  and 
Stirling,"  from  their  Text-Book  of  Physiology  ; by  " Schieffer decker,"  from  Das 
Mikroskop,  by  "Behrens,  Kossel,  and  Schieffer decker. 


PRACTICAL  HISTOLOGY 


PART  I. 


I.— APPARATUS  REQUIRED. 

The  student  of  Practical  Histology  must  be  provided  with  the 
following  apparatus: — 

1.  A Compound  Achromatic  Microscope  capable  of  magnify- 
ing from  about  50  to  300  or  450  diameters  linear. 

2.  Glass  Slides. — The  most  convenient  size  is  3 inches  by  i 
inch  (or  76  x 26  mm.),  with  ground  edges  made  of  the  best 
flatted  crown-glass.  About  two  gross  will  be  required  (fig.  i). 


76X26MS 

Fig.  t.— English  form  of  Slide. 

It  is  convenient  to  have  two  dozen  or  so  of  a larger  size,  3 inches 
by  1 1 inches. 

3.  Cover-Glasses. — Some  are  square  and  others  circular  (fig.  2). 


Fig.  2. — Square  and  Round  Cover-Glasses,  showing  the  most  convenient  sizes. 

The  student  should  be  provided  with  both  sorts.  Keep  the  square 
ones  for  balsam  preparations,  and  the  circles  for  those  that  require 
2 


18M5i 

2 


PRACTICAL  HISTOLOGY. 


ringing,  ^.e.,  those  mounted  in  glycerine  or  Farrant’s  solution. 
Only  extra-thin  covers  (or  those  sold  as  I^o.  i)  should  be  used. 
Jt  is  well  to  measure  the  thickness  of  the  covers,  and  to  use  for 
mounting  only  those  that  are  less  than  .006  inch  in  thickness. 

Get  half-an-ounce 
of  |-inch  circles, 
and  the  same 
weight  of  |-inch 
squares.  It  is  con- 
venient to  have 
a few  circles  and 
squares  somewhat 
larger,  viz.,  i inch 
and  I J inch  in  dia- 
meter, for  mount- 
ing i3articularly 
large  sections. 
When  a large  num- 
ber of  sections  are 
mounted  under 
one  cover-glass,  as 
have  oblong  cover-glasses  of  a 


Fig  3. — Pinewood  Cabinet  to  hold  Sixty  Slides. 


in  serial  preparations,  it  is  well 
still  larger  size. 


to 


4.  A Wooden  Cabi- 
net, fitted  with  trays 
for  holding  the  mounted 
specimens.  It  should  be 
capable  of  holding  at 
least  60  slides,  and  the 
slides  should  lie  on  the 
flat.  A convenient  form 
is  shown  in  fig.  3.  Some 
prefer  the  flat  com- 
pressed paper  trays 
shown  in  fig.  4. 

5.  Two  Mounted 
Needles  in  handles. 
The  student  can  easily 
make  these  liimself.  Fix 
a sewing-needle  into  the 
end  of  a pen-holder, 

allowing  only  about  ^ inch  of  the  needle  to  project.  The  needles 
should  always  he  kept  bright  and  polished.  A convenient  form  is 
to  fix  a sewing-needle  into  a strong  wooden  ‘‘  crochet  ” needle. 

6.  A Dissecting  Case,  hut  failing  that,  a pair  of  strong  forceps, 


Fig.  4. — Paper  Tray  to  hold  Slides. 


APPARATUS  REQUIRED. 


3 


and  also  a finer  pair,  the  latter  with  long  narrow  points ; a stout 
and  a fine  pair  of  scissors,  and  a scalpel.  It  is  well  to  have  a 
straight  pair  of  scissors,  and  also  a pair  curved  on  the  flat  (fig.  5.) 
It  is  convenient  to  have  a pair 
of  forceps  like  those  shown  in 
fig.  6 for  lifting  and  applying 
a cover-glass  to  a preparation, 
or  like  those  in  fig.  7 for  hold- 
ing a cover-glass  on  which  a thin 
film  with  bacteria  is  spread. 

7.  Camel-Hair  Brushes,  at 
least  two,  the  smaller  crow-size, 
and  one  somewhat  larger. 

8.  A Razor,  w^hich  is  not  to 
be  hollow-ground.  It  must  be 
kept  very  sharp,  and  stropped 
frequently.  It  is  better  to  have 
one  ground  flat  on  one  side. 

9.  Watch-Glasses. — Instead 
of  the  ordinary-sized  glasses, 
the  student  should  provide 
himself  with  at  least  four  3 
inches  in  diameter. 

10.  A Section-Lifter. — This  may  be  made  by  beating  out  the  end 
of  a piece  of  copper  wire  inch  thick)  until  a thin  plate  is  formed. 


Pig.  6.— Cover-Glass  Lifter. 


The  plate  is  then  bent  at  an  angle  to  the  stem.  It  is  better,  how- 
ever, to  purchase  one  made  of  German  silver  (fig.  8). 

11.  Drawing  Materials. — As  great  importance  is  attached  in 


Pig.  7.— Cornet's  Cover-Glass  Porceps. 


this  laboratory  and  in  this  course  to  making  drawings  of  the 
microscopic  objects,  each  student  must  provide  himself  with  a 
draivmg-hook — a quarto,  with  unruled  paper,  and  containing  150 
pages  or  thereby,  is  sufficient.  Suitable  draidng-pencils^  including 


4 


PRACTICAL  HISTOLOGY. 


an  H.B.,  and  a harder  one,  e.g.,  H.H.H.  ; both  must  be  of  a good 
quality  of  lead.  After  a sketch  has  been  made  in  pencil,  the 
sketches  should  be  coloured.  This  may  be  done  either  with 
coloured  pencils  or  water-colours.  The  latter  are  greatly  to 
be  preferred. 

12.  Slips  of  white  bibulous  paper,  3 inches  by  inch, 

Bto  soak  up  any  superfluous  fluid,  and  to  be  used  for  irriga- 
tion. For  irrigation  purposes  use  small  triangular  slips. 

13.  Small  glass  pipettes,  which  the  student  should 
make  for  himself  by  heating  in  a gas-flame  and  drawing 
out  a piece  of  narrow  glass-tubing  at  two  places,  close  to 
each  other,  leaving  a small  part  of  the  tube  of  the  original 
width,  which  acts  as  the  bulb  of  the  pipette.  Several  may 
be  made  at  a time,  and  their  capillary  ends  sealed  in  the 
flame,  and  kept  until  they  are  required. 

14.  A pair  of  narrow  glass  rods  drawn  to  a point  to 
tease  tissues  in  such  metallic  solutions  as  gold  chloride  or 
- silver  nitrate,  which  act  on  metallic  instruments. 

15.  Labels  for  the  slides. — It  is  well  to  have  a large 
number  of  pieces  of  paper  cut,  3 inches  by  i inch,  as 
temporary  labels,  on  which  is  written  the  name  of  the 
preparation.  Each  label  is  placed  under  its  a[>propriate 
i * slide  in  a tray.  These  labels  are  merely  temporary.  This 
H is  specially  desirable  where  the  slides  have  to  be  “ ringed, 
M as  in  this  process  a permanent  label  is  apt  to  be  displaced 
or  destroyed.  In  the  case  of  balsam  preparations,  they 
may  be  labelled  at  once  with  the  small  square  permanent 
labels.  In  every  case  the  preparation  should  be  labelled. 
Fig.  8.—  label  should  bear  not  only  the  name  of  the  tissue 

Lifter,  or  organ,  but  the  direction  of  the  section  and  the  medium 
in  which  it  is  mounted,  and,  if  desired,  the  date  of  mount- 
ing. Labels  are  now  printed  so  cheaply,  that  for  half-a-crown  a 
student  can  have  a thousand  labels  printed  with  his  own  name. 

16.  Reagents. — The  student  should  also  be  provided  with  the 
following  reagents,  placed  in  a small  wooden  framework  on  the 
work-table.  Only  those  reagents  that  are  most  frequently  used 
need  be  provided  for  in  the  framework  ; the  others  can  be  supplied 
as  required. 

Small  bottles — two  ounces  or  thereby — not  too  tall,  and  provided 
with  a glass  rod,  are  necessary.  The  glass  rod  has  a bulge  at  the 
junction  of  its  upper  and  middle  thirds,  and  this  bulge  prevents  it 
from  falling  into  the  bottle,  and,  at  the  same  time,  acts  as  stopper 
for  the  bottle.  Failing  this,  a piece  of  glass  rod  passed  through  the 
cork  will  answer  the  purpose. 

(1.)  Normal  Saline,  or  *6  per  cent,  salt  solution.  Dissolve  6 


THE  MICROSCOPE  AND  ITS  ACCESSORIES.  5 

grms.  of  pure  common  salt  in  looo  cc.  of  water.  As  this  fluid  is 
apt  to  undergo  change,  it  should  not  be  kept  too  long. 

(2.)  Glycerine  (either  pure  or  equal  parts  of  glycerine  and  water). 

(3.)  Balsam,  either  Canada  balsam  or  dammar  (p.  85). 

(4.)  Farrant’s  Solution  (p.  85). 

(5.)  Dilute  Acetic  Acid  (2  per  cent.). 

(6.)  Hsematoxylin  Solution  (p.  68). 

(7.)  Picro-Carmine  (p.  66). 

(8.)  Clove-Oil  or  Xylol.  — ^This  should  be  provided  with  a small 
brush  fixed  on  the  end  of  a wooden  rod  perforating  the  cork. 

17.  Other  Apparatus  is  required,  but  in  a well-equipped 
laboratory  special  articles  are  supplied  as  required ; they  are  referred 
to  in  the  context.  They  include  a dissecting  microscope,  photophore, 
mounting  block,  warm  stage,  eye-piece  micrometer,  lamp,  turntable, 
polarising  apparatus,  camera  lucida,  <fec.,  &c. 


II.— THE  MICROSCOPE  AND  ITS  ACCESSORIES. 

1.  An  account  of  the  optical  principles  on  which  the  microscope 
is  constructed  is  ])urposely  omitted.  The  compound  microscope 
consists  of  a stand  fixed  to  a heavy,  usually  horse-shoe  shaped,  foot. 
The  stand  (fig.  9)  carries  a stage  to  support  the  microscopic 
preparation,  the  mirror  or  arrangement  for  illuminating  the  object, 
together  with  the  body  tube  ; the  latter  consists  of  a long  brass  tube, 
or  one  tube  telescoped  into  another.  To  the  lower  end  of  this  tube 
is  fixed  a combination  of  lenses,  constituting  the  lens  or  objective, 
while  at  its  upper  end  is  the  eye-piece. 

2.  The  tube  is  blackened  inside,  and  to  its  lower  end  is  screwed 
the  objective,  consisting  usually  of  several  lenses  screwed  together. 
By  means  of  it  a magnified  inverted  aerial  image  is  produced  in  the 
body  of  the  tube.  The  lenses  on  the  objective  should  not  be 
unscrewed.  At  least  two  objectives  are  required — a low  power 
and  a high  power. 

At  the  upper  end  of  the  tube  is  the  ocular  or  eye-piece,  com- 
posed of  two  plano-convex  lenses,  the  one  next  the  eye  of  the 
observer  being  called  the  eye-glass^  the  lower  one  the  jidd-glass. 
The  two  lenses,  with  their  convex  surfaces  downwards,  are  fixed  in 
a brass  tube  which  slips  into  the  upper  end  of  the  tube  of  the 
microscope. 

3.  The  tube  itself  is  supported  in  a vertical  position  on  the  stand, 
so  that  it  can  be  moved  upwards  and  downwards  vertically,  to  bring 
the  objective  near  to  the  object,  and  thus  bring  the  latter  clearly 
into  focus.  This  arrangement  is  termed  the  adjustment.  The 


6 


PRACTICAL  HISTOLOGY. 


Body  tube. 


mode  in  which  this  is  accomplished  varies.  In  the  cheaper 
microscopes  the  tube  of  the  microscope  is  moved  inside  another 

tube  fixed  to  the 
Eye-piece.  stand  (fig.  9).  This 

is  done  by  means  of 
the  hand,  while  in 
the  more  expensive 
microscopes  there  is  a 
rack-and-pinion  move- 
ment for  raising  or 
depressing  the  tube 
(fig.  10). 

Usually  there  are 
two  adjustments — one 
the  coarse  adjust- 
ment, whether  it  be 
by  rotating  one  tube 
inside  the  other  or 
by  a rack-and-pinion 
movement ; it  is  used 
to  bring  the  outlines 
of  the  object  dimly 
into  focus.  The  other, 
the  fine  adjustment, 
is  a fine  screw,  usually 
placed  at  the  upper 
and  back  part  of  the 
pillar  of  the  stand  of 
the  microscope.  By 
it  the  object  is  brought 
accurately  into  focus. 

4.  The  stand  is  pro- 
vided with  a hori- 
zontal solid  table  or 
stage,  placed  at  a con- 
venient height,  and 
on  which  the  object 
to  be  examined  is 
placed.  The  stage 
consists  of  a flat  plate 
of  brass  blackened  on 
its  under  surface,  or 
There  are  two  clips 


stage. 


Fig.  9.— Leitz  s Microscope,  No.  V. 


of  a glass  plate  fi.xed  on  a black  ground, 
which  are  used  for  fixing  a preparation  in  a definite  position  on  the 
stage.  It  is  perforated  by  a circular  aperture  in  its  centre,  into 


THE  MICROSCOPE  AND  ITS  ACCESSORIES. 


7 


which  can  be  fitted  diaphragms  of  different  sizes,  or  a blackened 
circular  brass  plate  with  holes  of  different  sizes — from  a pinhole  to 
half  an  inch— rotates  under  the  stage,  so  that  the  desired  size  of  hole 
— or  diaphragm — can  be  brought  under  the  central  aperture  in  the 
stage.  The  aperture  in  the  stage  must  not  be  too  small ; it  should 
be  sufficiently  large  to  enable  a section  of  the  spinal  cord  to  be 
seen  as  a whole.  A small  aperture  is  used  with  high  powers  and  a 
large  aperture  with  low  powers.  In  the  more  expensive,  and  in 
some  of  the  cheaper  microscopes  also,  the  stand  is  provided 
with  a joint,  so  that  the  microscope  can  be  inclined  as  shown  in 

5.  Illumination  of  the  Object. — Under  the  stage  is  placed  a 
mirror,  movable  in  all  directions,  and  which  is  usually  provided 
with  a flat  and  a concave  surface.  When  it  is  available,  diffuse 
light — never  direct  sunlight — reflected  from  a white  cloud,  and  a 
northerly  exposure  are  to  be  preferred.  For  ordinary  illumination 
the  concave  side  of  the  mirror  is  used.  The  light  is  reflected  from 
it,  and  is  transmitted  through  the  hole  in  the  stage,  the  object  on 
the  stage,  and  the  tube  of  the  'microscope,  to  the  eye  of  the 
observer.  The  flat  mirror  is  used  along  with  a sub-stage  condenser 
(§  lo). 

6.  Direct  and  Oblique  Illumination. — When  the  light  is  re- 

flected from  the  concave  mirror,  it  strikes  the  object  nearly  verti- 
cally ; this  is  called  direct  or  central  illumination.  But  sometimes 
it  is  of  importance  to  detect  very  fine  variations  on  the  surface  of 
the  object;  then  for  this  purpose  oblique  illumination  is  practised. 
This  may  be  done  by  tilting  the  mirror  slightly,  so  that  the  rays 
of  light  fall  somewhat  obliquely  on  the  object.  In  this  case  there 
must  be  no  small  diaphragm  in  or  under  the  stage.  This  may  also 
be  done  in  the  more  expensive  microscopes  by  introducing  a 
diaphragm  which  permits  light  to  pass  only  at  its  sides,  its 
centre  being  blocked.  This  is  known  as  a central  stop-dia- 
phragm, which  shuts  off  all  the  axial,  and  transmits  only  the 
marginal  rays,  causing  what  is  called  dark-ground  illumination 
(fig.  12).  ^ 

7.  The  Diaphragm,  of  which  there  are  two  forms  in  common 
use.  The  most  common  form  is  a blackened  metallic  plate — disc 
diaphragm — perforated  with  holes  of  different  sizes,  placed  under 
the  stage,  and  so  arranged  as  to  rotate  on  a pivot.  The  edge  of  the 
plate  usually  projects  a little  beyond  the  stage,  so  that  it  can  be 
readily  rotated  by  the  finger,  so  as  to  bring  the  appropriate  aperture 
under  the  hole  in  the  stage.  The  diaphragm  is  usually  provided 
with  a slightly  projecting  pin,  which  gives  a click  when  the  hole  in 
the  diaphragm  is  exactly  centred.  Another  form — cylindrical 
diaphragm — consists  of  a small  brass  cylinder,  into  which  can  be 


8 


PRACTICAL  HISTOLOGY. 


fitted  small  diaphragms  perforated  by  apertures  of  different  sizes 
(figs.  lo,  12).  The  cylinder,  with  its  diaphragm,  is  fitted  into  a 
slot  under  the  stage.  These  cylinder  diaphragms  should  he  so 
arranged  as  to  he  easily  changed. 

Perhaps  the  most  convenient  form  of  all  is  the  iris  diaphragm^ 
which  can  he  adapted  to  any  of  the  larger  microscopes  (fig.  14), 
and  in  which  any  size  of  aj)erture  desired  is  obtained  by  turning  a 
small  milled  head.  The  new  form  (fig.  ii),  as  made  by  English 

makers,  Zeiss,  and  others, 
and  also  by  Leitz,  is 
an  admirable  substitute 
for  interchangeable  dia- 
phragms, as  by  it  the 
aperture  can  be  readily 
increased  or  diminished. 
The  smallest  aperture  is 
about  0.5  mm.,  and  the 
largest  equal  to  the  full 
aperture  of  the  con- 
densing system. 

The  management  of 
the  diaphragm  is  most 
important  in  order  to 
obtain  distinct  definition 
of  an  object.  The  one 
general  rule — but  one 
which  is  very  frequently 
neglected  in  practice  by 
the  student — is  when 
employing  a low  power 
to  use  a large  aperture, 
I and  when  employing  a 
high  power,  a small  or 
medium  aperture  of  the 
diaphragm. 

When  one  wishes  to 
e a brightly  col- 
oured object  with  a 
homogeneous  immersion  lens — the  object  lying  in  the  tissues,  such 
as  stained  fungi  or  mitotic  figures — then  remove  the  diaphragm  and 
allow  a flood  of  light  to  reach  the  sub-stage  condenser,  which  is 
absolutely  necessary  in  this  case. 

If,  however,  it  be  desired  to  see  certain  peculiarities  of  structure 
— as  in  bacteria — on  a black  background,  then  use  a central  stop- 
diaphragm,  one  with  a central  stop  (fig.  12),  always  with  an 


^IG.  10.— Zeiss’s  Large  Jointed  Stand,  fitted  with  draw- 
tube,  rack  and  pinion  for  the  coarse  adjustment,  a 
double  nose-piece,  and  cylindrical  diaphragm.  ODServ 


THE  MICROSCOPE  AND  ITS  ACCESSORIES. 


9 


Abbe’s  condenser.  The  object  is  then  seen  brilliantly  illuminated 
on  a black  background. 

8.  Objectives. — For  ordinary  work  every  microscope  requires  to 
have  two  objectives  of  different  magnifying  powers ; one  of  these, 
when  used  with  an  ordinary  ocular,  should  magnify  about  60-75 


Fig.  II, — Iris  Diaphragm,  showing  a small 
aperture. 


diameters  linear.  This  is  spoken  of  as  the  low  power,  and  in  these 
pages  is  indicated  by  the  letter  (L).  The  other  should  magnify 
from  350-400  diameters  linear,  and  is  called  the  liigh  power, 
indicated  by  the  letter  (H). 

If  an  English  make  of  lens  is  preferred,  let  them  be  an  “ inch  ” 
and  a “quarter  inch.”  The  term  “ one-inch objective ” has  no  direct 
relatioii  to  the  distance  between  the  object  and  the  lens,  but  indi- 
cates that  such  a lens  possesses  the  same  magnifying  power  as  a single 
lens  of  one-inch  focus.  Swift’s  new  high-angled  i-inch  lens  is  a 
very  good  low-power  lens.  If  Continental  lenses  be  preferred,  and 
if  Zeiss’s  be  selected,  let  them  be  A and  D,  equal  to  §-inch  and 
|-inch  respectively.  Zeiss  makes  lenses  AA  andDD  of  slightly  better 
quality,  which  cost  a few  shillings  more.  If  Leitz’s  lenses  be  pre- 
ferred, use  Nos.  3 and  7 ; and  if  Hartnack’s,  Nos.  3 and  7 — 3 being 
the  weaker  lens.  Crouch’s  lenses  are  excellent. 

Leitz  numbers  his  objectives  1-9,  and  Zeiss  A-F.  i and  A are  the 
weakest  objectives,  and  the  magnifying  power  increases  up  to  9 and  F. 

The  microscopes  of  Reichert  of  Vienna  are  also  excellent.  The 
stands  are  all  provided  with  a universal  thread  or  screw,  so  that  the 
lenses  of  Zeiss,  of  English  makers,  or  of  others  may  be  adapted  to 
them. 

For  certain  special  purposes  much  higher  powers  are  required, 
but  for  ordinary  work  these  lenses  are  sufficient. 

3 


lO 


PRACTICAL  HISTOLOGY. 


The  following  patterns  of  microscopes  are  to  be  commended  : — 

Leitz,  Stands  Nos.  Y.,  III.  17,  IV.  19,  V,  23,  with  objectives 
3 and  7. 

Zeiss,  Stands  Nos.  YL  and  YIT.,  with  objectives  C and  E. 

9.  Eye-Pieces  or  Oculars. — Two  eye-pieces  are  required,  one 
medium  length  and  the  other  shorter,  the  latter  being  the  more 
powerful.  The  two  most  useful  eye-pieces  are  Nos  II.  and  lY,  of 
foreign  makers  (of  Leitz,  however,  I.  and  III.),  or  A and  C of 
English  makers.  English  makers  speak  of  a deep  and  a shallow  eye- 
piece ; the  former  (lY.  or  is  shorter,  and  is  the  more  powerful ; 
the  latter  (II.  or  A)  is  longer,  and  is  a weaker  eye-piece. 

It  is  to  be  remembered  that  the  eye-piece  only  magnifies  the 
image  of  the  preparation  formed  by  the  objective,  and  does  not 
magnify  the  preparation  itself.  Hence  any  fault  in  the  lens,  and 
consequently  in  the  image  in  the  tube,  is  magnified  by  the  eye- 
piece. Moreover,  the  field  is  not  so  bright  as  with  a weaker  eye- 
piece. Hence  it  is  expedient  to  use  rather  a weak  eye-piece.  There 
is  an  exception  to  this  rule  in  the  case  of  apochromatic  lenses  (p.  13), 
which  yield  a magnification  of  the  object  by  means  of  special  eye- 
pieces, without  any  loss  in  the  brightness  or  sharpness  of  the  image. 

These  comprise  the  essential  parts  of  the  microscope,  but  if 
expense  be  no  objection,  the  stand  may  be  provided  with  a hinge- 
joint,  which  enables  the  microscope  to  be  inclined  at  any  angle  (figs. 
10,  14).  In  some  microscopes  the  tube  can  be  elongated  by  a draic- 
tuhe  (fig.  14),  which,  when  it  is  elongated,  increases  the  magnifying 
power  of  the  instrument.  It  is  well  to  have  the  draw-tube  with  a 
scale  engraved  on  it,  as  is  done  in  the  more  expensive  instruments. 

10.  Sub-Stage  Condenser. — When  working  with  high  powers,  this 
is  essential,  more  especially  in  connection  with  bacteriological  work. 
Abbe’s  condenser  is  by  far  the  most  convenient  form.  Fig.  14  shows 
one  of  the  more  expensive  stands  fitted  with  a sub-stage  condenser. 

The  essential  feature  of  Abbe’s  illuminating  apparatus  is  a con- 
denser system  of  very  short  focus,  which  collects  the  light  reflected 
by  the  mirror  into  a cone  of  rays  of  very  large  aperture  and  projects 
it  on  the  object  (fig.  13). 

The  cone  of  light  is  usually  reduced  by  diaphragms  of  suitable 
size,  or  by  means  of  an  iris  diaphragm.  By  means  of  the  rack-work 
the  diaphragm  can  be  placed  excentrically  and  oblique  illumination 
obtained.  This  apparatus  can  be  used  for  ordinary  work,  but  it  is 
specially  useful  for  the  investigation  of  bacteria.  The  rays  of  light 
from  the  condenser  are  brought  to  a focus  in  the  object,  so  that  an 
enormous  amount  of  light  is  concentrated  on  the  object.  The  angle 
of  aperture  of  Abbe’s  condenser  is  120°.  The  full  aperture  of  the 
illuminating  cone  of  rays  is  only  used  when  observing  deeply-stained 


THE  MICROSCOPE  AND  ITS  ACCESSORIES. 


-II 


bacteria  with  objectives  of  largo  aperture.  In  every  other  case  a 
diaphragm  of  suitable  size  is  introduced  so  as  to  diminish  the 
cone  of  light.  When  the  diaphragm  is  placed  excentrically,  oblique 
illumination  is  obtained,  while, 
with  a central-stop  diaphragm 
all  the  axial  rays  are  cut  off, 
and  dark-ground  illumination  is 
obtained. 

11.  Dry  and  Immersion 
Lenses. — By  the  term  dry  lenn 
is  meant  one  in  which  air  is 
the  medium  between  the  lens 
and  the  object,  or  at  least  the 
cover-glass  on  the  object.  In 
immersion  lenses  some  fluid 
intervenes  between  the  lowest 
lens  of  the  objective  and  the 
cover-glass  of  the  object,  and 
the  liquid  chosen  is  water,  or  a 
medium  of  higher  refractive 
index,  such  as  cedar-wood  oil, 
glycerine,  or  a mixture  of 
fennel  and  ricinus  oils.  These 
oils  have  nearly  the  same  re- 
fractive index  as  the  cover- 
glass.  In  virtue  of  their  greater  refractive  power,  these  liquids, 
especially  the  oils,  refract  more  of  the  rays  passing  through  the 
object,  and  cause  these  rays  of  light  to  enter  the  lens,  so  that  they 
increase  the  amount  of  light  transmitted  to  the  eye  of  the  observer. 
In  contrast  with  dry  lenses,  they  have  a larger  angle  of  aperture 
and  a greater  resolving  power,  and  are  employed  only  for  the 
highest  magnifying  powers.  Powers  above  ^ of  an  inch  should  be 
oil-immersion  lenses.  Oil-imraerdon  or  homogeneous  immersion 
lenses  are  to  be  preferred  to  water  ones.  The  oil-immersion  are 
fast  displacing  water- immersion  lenses. 

In  using  an  oil-immersion  lens,  by  means  of  a glass  rod,  place  a 
small  drop  of  thick  cedar-oil  on  the  cover-glass  and  a small  drop  on 
the  lowest  lens  of  the  objective,  and  slowly  depress  the  objective 
until  it  touches  the  cupola  of  the  drop,  and  focus.  Cedar-oil  has  a 
refractive  index  nearly  the  same  as  that  of  crown-glass,  so  that 
almost  all  the  rays  of  light  passing  through  the  object  reach  the  lens 
and  pass  up  the  tube  of  the  microscope.  Care  must  be  taken  that 
the  drop  of  oil  does  not  run  on  to  the  cement  of  the  preparation, 
else  it  will  dissolve  it.  It  is  better  to  use  marine  glue  to  seal  up 
the  preparation,  as  it  is  not  dissolved  by  cedar-oil,  A very  fine 


Fig.  13.— Abbe’s  Condenser,  as  made  by  Zeiss 
and  Swift.  (5.)  condenser  system.  Milled 
head  for  throwing  the  diaphragm  out  of 
centre  : {Sp.)  mirror. 


12 


PRACTICAL  HISTOLOGY. 


Fig.  14.— Leitz’s  Stand  la.,  with  a triple  nose-piece  and  Abbe’s  condenser 


THE  MICROSCOPE  AND  ITS  ACCESSORIES. 


13 


linen  cloth  moistened  with  benzene  may  be  used  to  remove  the  oil 
from  the  lens.  After  use,  the  cover-glass  must  be  wiped  dry,  as 
well  as  the  lens,  by  means  of  a clean  piece  of  wash-leather.  These 
lenses  are  very  good,  but  very  dear,  and  are  not  required  for  ordinary 
work.  Latterly,  instead  of  oil,  a mixture  of  glycerine  and  chloral 
hydrate  has  been  recommended.  The  cedar-oil  is  not  so  easily 
removed  from  the  cover-glass.  It  may  be  best  removed  by  a well- 
washed  linen  rag  moistened  with  benzene.  The  lens  itself  must  be 
most  carefully  cleaned.  A lens  moistened  with  glycerine  is  best 
cleaned  with  alcohol. 

12.  Angle  of  Aperture. — Lenses  may  be  of  narrow  or  wide  angle 
of  aperture.  The  angle  of  aperture  is  the  angle  formed  by  the 
outermost  rays  coming  from  a luminous  point  placed  in  the  focus  of 
an  object,  and  which  enter  not  only  the  lowest  lens  of  the  objective, 
but  pass  throughout  the  entire  system  of  the  lenses  of  the  objective. 
Lenses  with  a large  angle  of  aperture  (130°  and  upwards),  therefore, 
will  admit  more  light,  i.e.,  more  of  the  oblique  rays  will  enter  the 
system  of  lenses.  These  lenses  are  well  suited  for  *resolving  fine 
lines  on  the  surface  of  an  object,  such  as  the  strise  on  the  scales  of 
insects’  wings  and  the  markings  on  diatoms,  but  those  parts  of  the 
object  superficial  to  or  deeper  than  the  focus  are  not  sharply  defined. 
Such  a lens  is  said  to  have  greater  power  of  resolution.  For  ordi- 
nary histological  work,  a high-power  (350-400)  lens  of  medium 
(8o°-ioo°)  angular  aperture  is  to  be  preferred,  for  such  a lens  has 

pe7ietratin(f  ;powe7\  Ae.,  the  focal  plane  is  deeper,  so  that  with 
it  one  oan  see  with  tolerable  distinctness  parts  of  the  object  lying 
immediately  above  and  below  the  true  focus  of  the  lens.  It  is  to 
be  remembered  that  the  angle  of  aperture  has  nothing  to  do  with 
the  magnifying  power  of  the  lens. 

13.  Abbe’s  Apochromatic  Lenses. — These  are  dry  or  immersion, 
and  are  used  as  high-power  lenses  when  very  exact  definition  is 
required.  They  are  very  expensive,  and  are  constructed  of  a 
peculiar  kind  of  glass.  These  objectives  secure  the  union  of  three 
different  colours  of  the  spectrum  in  one  point  of  the  axis,  ^>.,  they 
remove  the  so-called  ‘‘secondary  spectrum,”  and  they  correct  the 
spherical  aberration  for  two  different  colours.  The  images  projected 
by  them  are  nearly  equally  sharp  with  all  the  colours  of  the 
spectrum.  As  there  is  a very  great  concentration  of  light  by  these 
objectives,  they  permit  of  the  use  of  very  high  eye-pieces,  thus  giving 
high  magnifying  power  with  relatively  long  focal  length.  The  natural 
colours  of  objects  are  reproduced  unaltered  by  these  objectives. 

Zeiss  has  constructed  a series  of  compensating  oculars  to  be 
used  with  these  lenses.  They  are  classified  as  i,  2,  4,  8,  12,  18, 
and  27,  according  to  their  magnifying  power.  The  eye-pieces  of 
extremely  low  power  are  designated — 


14 


PRACTICAL  HISTOLOGY. 


(i.)  Searchers,  which  reduce  to  its  lowest  limits  the  available 
magnification  with  each  objective,  thus  facilitating  the  preliminary 
examination  of  objects,  diminishing  the  labour  of  searching  for 
particular  parts  of  the  specimen.  Thus,  N’o.  i enables  an  objective 
to  be  employed  with  its  own  initial  magnifying  power,  i,e.,  as  if  it 
were  used  as  a simple  lens  without  an  ocular  {Zeiss), 


(ii. ) The  working  eye-jpieces  begin  with  a magnifying  power  of  4. 
The  most  useful  are  4 and  6.  Other  eye-pieces  are — 

(iii.)  Projection  eye-pieces ; but  they  do  not  concern  us  here. 

14.  In  selecting  a microscope,  there  are  many  points  to  be 
attended  to. 


THE  MICROSCOPE  AND  ITS  ACCESSORIES. 


15 


(A.)  Mechanical  Parts. — It  should  bo  quite  stable,  so  that  it 
cannot  be  readily  upset.  To  this  end  the  stand  should  be  solid,  and 
either  of  the  tripod  (fig.  15)  or  horse-shoe  pattern  (figs.  9,  10).  Very 
good  stands  of  the  tripod  pattern  are  made  by  Messrs.  James 
Swift  & Sons  (fig.  15)  and  Messrs.  Crouch.  Fig.  15  has  a glass  stage, 
the  body-tube  is  cloth-lined,  which  gives  a smooth  and  steady  action. 
The  stage  should  be  at  a convenient  height,  so  that  when  the  ulnar 
edge  of  the  left  hand  is  resting  on  the  table,  the  thumb  and  fore- 
finger of  the  same  hand  can  conveniently  grasp  and  move  the  slide  on 
the  stage.  The  stage  itself  should  be  a little  broader  than  the  length 


of  the  slide.  The  slide  can  bo  fixed  on  the  stage  by  means  of  two 
brass  clips,  which  are  fitted  into  holes  at 
the  two  posterior  angles  of  the  stage. 

The  pillar  of  the  microscope  may  be  fitted 
with  a joint  to  enable  the  instrument  to 
be  inclined,  if  desired;  but  of  course 
this  cannot  be  used  w’hen  fluids  are  being 
examined ; still  in  many  instances  it  is 
convenient  (figs.  10,  14).  Fig.  16  shows 
a convenient  form  made  by  Hartnack  of 
Potsdam,  and  one  very  extensively  used 
by  students. 

Itis  highly inconvenienttohave  to  screw 
and  unscrew  a lens  every  time  a change 
of  lens  is  required.  This  is  obviated  by 
using  a nose-piece  or  revolver  (figs.  10, 

14),  which  is  screwed  to  the  lower  end 
of  the  tube  of  the  microscope.  The  high 
and  low  powers  are  fitted  to  this  frame- 
work, and  can  be  rotated  under  the  tube 
as  they  are  required.  Fiose-pieces  are 
made  for  attaching  two  or  three  (figs.  10, 

14)  or  more  objectives,  and  they  can  be 
adapted  to  any  microscope. 

One  must  be  cautious  in  using  a nose-piece,  and  take  care  to  raise 
the  tube  high  enough  to  allow  the  objective  to  revolve  without  touch- 
ing the  cover-glass,  which  is  especially  apt  to  happen  on  using  a high 
power  after  a low  power,  the  lenses  not  being  of  the  same  length. 

Test  the  mechanical  parts  that  they  are  all  solid  and  work  well. 
Raise  and  lower  the  tube  by  means  of  the  fine  adjustment,  and  do 
this  to  the  full  extent  of  the  threads  on  the  screw,  noting  parti- 
cularly if  there  is  any  lateral  movement  of  the  tube  while  this  is 
being  done. 

(B.)  Optical  Parts. — See  that  all  the  parts  are  properly  centred  by 
looking  through  the  tube  after  removal  of  the  eye-piece.  Put  on 


Fig.  16. — Stand  III.  of  Hartnack, 
with  joint  and  condenser. 


i6 


PRACTICAL  HISTOLOGY. 


the  high  power ; use  a medium  eye-piece,  and  focus  a microscopic 
preparation ; any  thin  section  of  a tissue  will  do.  The  field  should 
be  large,  well  illuminated,  and  flat.  If  there  are  specks  in  it,  clean 
all  the  lenses  to  see  that  these  specks  are  not  due  to  dust  on  the 
lenses.  If  the  centre  of  a flat  object  in  the  field  does  not  come  into 
focus  at  the  same  time  as  the  periphery  of  the  object,  then  the  lens 
should  be  rejected  on  account  of  its  spherical  aberration.  If  coloured 
rings  appear  in  an  object,  then  the  lens  must  also  he  rejected,  as  it 
is  not  perfectly  achromatic.  The  definition  ought  to  be  sharp  and 
distinct. 

The  qualities  of  a good  lens  are  the  following : — 

The  definition  should  be  good,  the  correction  for  spherical 
and  chromatic  aberration  should  be  perfect.  The  outlines  of  objects 
should  be  sharply  defined  and  not  blurred,  and  there  should  be  no 
coloured  halos  or  fringes  round  the  object.  If  so,  the  chromatic 
aberration  of  the  lens  is  not  perfectly  corrected. 

Flatness  of  field,  t.e.,  all  parts  of  the  object  in  the  field  at  the  same 
time  should  be  seen  with  equal  distinctness,  and,  of  course,  this  can 
only  be  so  when  the  field  is  flat.  If  the  central  parts  are  sharply 
focussed,  the  peripheral  parts  not,  then  the  field  is  not  flat. 

Resolving  power,  which  depends  on  the  angle  of  aperture.  This 
is  the  power  to  render  visible  surface  markings,  superficial  lines,  or 
structural  details.  It  is  better  to  have  a lens  of  large  angle  of 
aperture  for  this  purpose. 

Penetrating  power,  or  the  power  to  see  objects  in  several  planes 
in  the  same  preparation  at  the  same  time.  It  represents  the  focal 
depth  of  the  lens  and  its  power  of  focussing  images  from  different 
planes  in  the  vertical  range,  l^arrow  angled  lenses  are  more  suit- 
able for  this  purpose. 

Working  Distance,  ^.e.,  the  distance  between  the  lens  and  the 
object.  It  has  no  direct  relation  to  the  focal  length  of  a lens. 
Wide  angle  lenses  have  a shorter  working  distance  than  narrow  angle 
lenses.  A good  lens  should  combine  all  the  foregoing  qualities,  but 
of  these  qualities  definition  is  all  important. 

15.  Drawing  of  Microscopic  Objects. — In  this  laboratory  every 
student  is  required  to  make  sketches  of  his  preparations.  This  is 
of  the  utmost  importance,  not  only  from  the  point  of  view  of  the 
student,  but  also  of  the  teacher. 

(A.)  Freehand  Sketching. — The  student  must  provide  himself 
with  a drawing-hoolc  (p.  3)  and  with  suitable  pencils  (H.B.  and 
H.H.H.),  and  also  with  either  coloured  crayons  or  water-colours. 
It  is  far  more  important  that  an  outline  sketch  in  pencil  be  made 
accurately  portraying  the  shape  of  the  object,  than  that  an  indifferent 
sketch  should  be  covered  with  a smear  of  colour.  It  is  astonishing 
how  some  students,  after  protesting  that  they*  cannot  “ drawq” 


THE  MICROSCOPE  AND  ITS  ACCESSORIES. 


17 


succeed  in  delineating  their  preparations  when  they  have  given  the 
matter  a fair  trial. 

(B.)  Various  Forms  of  Camera  Lucida. — Sometimes,  however, 
a drawing  has  to  be  done  to  scale,  or  its  outlines  and  details 
accurately  portrayed.  There  are  many 
devices  for  this  purpose.  Some  ol 
these  instruments  are  by  no  means 
easy  to  work  with,  but'  they  are 
excellent  for  tracing  the  outlines  of 
objects  and  showing  the  exact  rela- 
tion of  one  part  to  another,  and  the 
relative  size  of  the  parts  of  an  object. 

(i.)  Zeiss's  Camera  Lucida  (fig. 

17). — collar  (c)  with  a vertical  rod 
attached  (a),  carrying  a bar  (h)  which 
bears  the  prism  or  camera  (A").  The 
collar  is  slipped  over  the  tube  of  the 
microscope  and  the  eye-piece  inserted. 

Focus  the  object,  and  rotate  the  prism 

until  it  is  above  the  eye-piece,  the  bars ; (/r.) camera, 
prism  covers  one-half  of  the  latter.  ^ i i i • 

The  drawing-board  is  inclined  at  an  angle  of  20  , and  in  looking 
down  the  microscope  the  paper  and  the  image  of  the  object  are 

seen  simultaneously.  . i 1 v • 

(ii.)  Camera  Lucida  (Abbe).— This  apparatus  is  made  by  Zeiss 
(fig.  18).  The  apparatus  is  screwed  to  the  tube  of  the  microscope. 


The  direction  and  reflection  of  the  rays  from  the  object  and  those 
from  the  paper  are  shown  in  the  figure.  In  all  camera-lucida 
drawinexs  it  is  important  to  regulate  the  brightness  of  the  illumination 
® B 


i8 


PRACTICAL  HISTOLOGY. 


of  the  paper ; this  is  effected  in  this  instrument  by  smoke-tinted 
glasses,  which  fit  into  the  prism  mounting.  In  other  cases  this  is 
done  by  means  of  a blackened  cardboard  shade. 

(iii.)  Chevalier’s  Camera  Lucida  (fig.  19). — The  microscope 
remains  erect,  the  eye-piece  is  removed,  and  in  its  place  is  inserted 


rays  have  to  pass  through  what  is  practically  an  eye- piece  of  a 
microscope,  and  reach  a small  prism  at  the  free  end  of  the  hori- 
zontal part  of  the  instrument.  This  prism  is  so  arranged  that  the 
rays  coming  from  the  object  are  reflected  into  the  eye  of  an  observer, 
who,  on  looking  through  the  prism,  sees  an  image  of  the  object  upon 
the  sheet  of  paper  on  the  table.  The  observer  looks  through  this 
prism,  but  he  must  so  adjust  his  position  as  to  bring  one-half  of  the 
pupil  over  the  prism,  and  thus  with  one-half  of  the  pupil  view  the 
object  on  the  paper,  while  the  other  half  of  the  pupil  receives  the 
rays  of  light  coming  from  the  pencil  and  the  paper.  The  distance 
between  the  eye  of  the  observer  and  the  paper  is  about  10  inches. 
It  is  by  no  means  so  easy  to  sketch  the  outline  of  an  object  with 
this  camera.  The  eye  should  be  protected  from  other  rays  of  light 
by  means  of  a blackened  shade.  The  difficulty  encountered  by 
the  student  is  to  see  the  object  and  the  point  of  the  pencil  at  the 
same  time. 

(iv.)  Camera  of  Malassez  (fig.  20). — This  consists  of  two  prisms; 
one,  the  ocular  prism,  is  fixed,  while  the  second  and  larger  can  be 
moved  round  a horizontal  axis.  This  enables  the  instrument  to  be 
used  in  two  different  positions  of  the  microscope.  If  one  is 
examining  a fluid,  the  microscope  is  erect  and  not  inclined.  The 
instrument  is  fixed  to  the  upper  part  of  the  tube  of  the  microscope. 
Slip  the  collar  over  the  tube  and  insert  the  eye-piece.  The  image 
is  projected  on  the  table  to  the  right  of  the  microscope ; but  the 
paper  must  be  placed  at  an  angle,  t.e.,  on  an  inclined  plane,  horizontal 
to  the  direction  of  the  rays  from  the  camera.  This  is  necessary  to 
avoid  one  side  of  the  figure  being  larger  than  the  other.  If  it  be 


Fig.  19. — Camera  Lucida  of  Chevalier  or 
Oberhauser. 


the  tube  of  the  camera.  Place 
a sheet  of  white  paper  at  the 
side  of  the  microscope,  and 
directly  under  the  horizontal 
part  01  the  camera.  The  in- 
strument itself  consists  of 
two  tubes  at  right  angles  to 
each  other,  and  at  the  angle 
is  a prism,  which  reflects  at 
a right  angle  in  a horizontal 
direction  the  light  coming 
vertically  from  an  object  on 
the  stage.  These  horizontal 


THE  MICROSCOPE  AND  ITS  ACCESSORIES. 


19 


desired  to  incline  the  microscope  at  an  angle  of  45°  (fig.  20),  the 
camera  is  so  placed  that  an  image  is  thrown  on  paper  placed  behind 
the  foot  of  the  microscope,  the  prism  being  turned  to  an  angle  of 
45°.  This  camera  is  particularly 
easy  to  work  with.  It  is  some- 
times important  not  to  have  too 
much  light  on  the  paper ; this  is 
avoided  by  using  the  plane  side  of 
the  mirror. 

16.  Magnifying  Power  of  a 
Microscope. — This  will  vary  with 
the  objective  and  ocular  used,  and 
also  with  the  length  of  the  draw- 
tube.  The  magnification  is  usually 
determined  when  an  image  is  seen 
at  the  range  of  normal  distinct 
vision,  i.e.^  10  inches  or  25  centi- 
metres. As  this  is  about  the 
height  of  a Hartnack’s  stand  or 
Zeiss’s  stand,  supposing  these 
forms  of  instrument  to  be  used, 
place  a sheet  of  white  paper  on 
the  table.  Let  the  microscope  be  upright,  with  ocular  and  lens  in 
place.  Supposing  we  use  a No.  7 objective  and  No.  3 ocular. 
Begin  with  the  draw-tube  in,  place  a stage-micrometer  on  the  stage 
of  the  microscope,  and  focus  the  scale  upon  it.  The  stage-micro- 
meter is  like  a glass  slide  with  a fine  scale  engraved  upon  it.  The 
English  ones  are  generally  subdivided  into  thousandths  of  an  inch, 
and  the  Continental  ones  into  hundredths  of  a millimetre.  Look 
through  the  microscope,  but  keep  both  eyes  open,  and  part  of  the 
scale  will  be  seen  on  the  white  ])aper.  With  a pencil  mark  off  say 
ten  interspaces,  or  use  a pair  of  compasses  to  measure  this  distance. 
Measure  off  this  distance  on  a millimetre  scale,  and  suppose  the 
distance  thus  measured  be  28  millimetres.  This  means  that  .1 
millimetre  has  been  magnified  to  appear  equal  to  28  millimetres, 
^.e.,  280  times.  The  magnifying  power  for  other  combinations  of 
lenses  should  be  determined,  and  a table  made  for  future  reference 
and  use. 

Magnifying  Power. — This  may  be  increased — 


Tig.  20.— Malassez’s  Camera  placed  at  a 
variable  angle. 


(a)  By  using  a higher  objective. 

(/))  By  using  a higher  eye-piece. 

(c)  By  pulling  out  the  draw-tube  and  increasing  the  distance 
between  the  objective  and  the  eye-piece. 

Construct  a table — according  to  the  following  scheme— of  the 


20 


PRACTICAL  HISTOLOGY. 


magnifying  powers  of  the  different  combinations  of  the  objective 
and  ocular  supplied  with  your  microscope  : — 


Objective. 

Ocular. 

Shallow,  or  A or  2. 

1 

Deep,  or  C or  4. 

1 

-P,  1.  1 • r Half-inch  or  A 

raw-  u e in  | Qne-sixth  inch  or  D 

, , . i Half-inch  or  A 

Draw-tube  out  | Que-sixtl.  inch  or  D 

17.  Measurement  of  the  Size  of  a Microscopic  Object. — Sup- 
pose a red  blood-corpuscle  is  to  be  measured.  Keep  both  eyes 
open  ; with  one  look  down  the  tube  of  the  microscope,  and  the 
other  eye  will  see  an  image  of  the  corpuscles  on  a sheet  of  white 
paper  placed  beside  the  microscope.  With  a pencil  mark  off  the 
outlines  of  a corpuscle,  or  a sketch  may  be  made  of  one  with  a 
camera  lucida.  Remove  the  preparation,  and  for  it  substitute  a 
stage-micrometer,  leaving  all  the  other  parts,  microscope  and  paper, 
as  they  were.  On  looking  down  the  tube  of  the  microscope,  the 
lines  on  the  micrometer  scale  are  seen.  Make  a drawing  of  these 
lines.  This  may  be  kept  for  future  use,  but  on  the  scale  should  be 
noted  the  combination  of  ocular  and  objective,  and  the  extent  to 
which  the  draw-tube  has  been  elongated.  The  distance  between 
the  lines  on  the  micrometer  scale  being  known,  say  y-J^th  of  a 
millimetre,  it  is  easy  to  calculate  what  part  of  the  distance  between 
any  two  lines  corresponds  to  the  size  of  the  corpuscle. 

The  most  expeditious  plan  is  to  use  an  eye-piece  micrometer. 
A circular  flat  piece  of  glass,  with  a scale  ruled  on  it  (fig.  21),  is  in- 
serted in  the  ocular  between  the  field-glass  (FG) 
and  the  eye-glass  (EG)  (fig.  22).  With  this  eye- 
piece focus  the  scale  on  a stage-micrometer — its 
markings  must  be  parallel  to  these  in  the  eye- 
piece— and  count  the  number  of  divisions  of  the 
latter  that  correspond  to  one  of  the  former.  Tliis 
must  be  determined  for  each  combination  of  lenses 
with  a known  length  of  tube.  Suppose  the  stage- 
micrometer  to  be  divided  into  y^^ths  of  a milli- 
metre, and  that  one  of  these  divisions  corresponded 
to  three  of  those  in  the  ocular,  then  each  of  the 
spaces  in  the  ocular  micrometer  is  equal  to  y Jotli  of  a millimetre, 
or  .0033  millimetre. 

The  histological  unit  of  measurement  is  1 000th  part  of  a milli- 


Fia.  21. — Scale  intro- 
duced into  Eye- 
piece Micrometer 
{Zeiss). 


THE  MICROSCOPE  AND  ITS  ACCESSORIES. 


21 


metre  or  micro-millimetre,  expressed  shortly  as  a micron,  or  by 
the  Greek  letter  /x.  Thus,  each  space  of  the  ocular  micrometer  is 
equal  to  3-3  /x. 

With  these  data  it  is  easy  to  estimate  the  size  of  any  object.  The 
object  is  placed  on  the  stage  and  focussed,  always,  of  course,  with 
the  same  combination  of  lenses  and  ocular.  If  other  lenses  be  used, 
the  value  of  the  ocular  divisions  must  be  determined  for  this  particular 
combination.  Once  determined,  they  can  be  noted  for  future  use. 

18.  Artificial  Illumination. — White  daylight  is,  of  course,  to  be 
preferred,  but  it  is  not  always  available.  When  artificial  light  has 
to  be  used,  a gas  flame,  e.g.^  an  Argand  burner,  or  other  artificial 
light,  may  be  used.  An  ordinary  paraffin  lamp  with  a flat  wick  and 
arranged  to  burn  steadily  does  very  well.  An  unsteady  flame  is 


Fia.  23. — Microscope  Lamp. 


very  injurious  to  the  eyes.  It  requires  some  care  to  find  the  exact 
distance  at  which  the  lamp  should  be  placed  from  the  mirror.  If 
very  intense  light  be  required  to  examine  a small  part  of  a pre- 
paration, turn  the  wick  edge  on  to  the  mirror.  Between  it  and  the 
mirror  place  a screen  of  white  paper.  If  the  direct  rays  from  a 
lamp  are  used,  to  correct  the  yellow  rays  place  a sheet  of  pale  blue 
glass,  or  a glass  globe  containing  a weak  solution  of  ammonio- 
sulphate  of  copper,  between  the  light  and  the  mirror,  or  a thin  blue 
glass  may  be  placed  on  the  stage  under  the  preparation.  A paraffin- 
oil  lamp  may  be  used.  Fig.  23  shows  a cheap  paraffin  lamp  sold  by 
Swift  & Sons.  A remarkably  good  light  is  obtained  from  the 


22 


PRACTICAL  HISTOLOGY. 


Welsbach  gas-burner,  the  rays  being  transmitted  through  a large 
globe  containing  a dilute  blue-coloured  solution  of  ammonio-sulphate 


Fig.  24. — Dissecting  Microscope  by  Vorick. 


of  copper.  The  lamp  recently  constructed  by  Kochs-Wolz  of  Bonn 
consists  of  a petroleum  or  gas  flame,  covered  by  an  opaque  chimney 


provided  with  a reflector,  which  directs  the  rays  of  light  through  a 
hole  in  the  chimney.  Into  the  hole  in  the  chimney  is  fitted  a glass 


THE  MICROSCOPE  AND  ITS  ACCESSORIES. 


23 


rod,  which,  bent  in  a gentle  curve,  conducts  the  light  from  the 
flame  to  the  under  surface  of  the  stage,  the  mirror  in  this  case  not 
being  used. 

Perhaps  the  most  useful  lamp  is  that  known  as  the  albo-carbon 
light.  It  is  the  one  I am  in  the  habit  of  using  when — especially 
in  winter — good  daylight  is  not  available.  Ordinary  gas  is  used, 
but  the  gas  has  to  traverse  a chamber  containing  naphthaline  before 
it  reaches  the  burner.  A very  white  light  is  thus  obtained,  and 
can  be  used  without  the  intervention  of  blue  glass  or  a blue  copper 
solution. 

If  expense  be  no  bar,  then  a small  incandescent  electric  light  is 
most  useful. 

19.  Dissecting  Microscope  (figs.  24,  25). — This  is  very  useful. 
The  lenses  usually  employed  magnify  from  5 to  20  times  linear, 
and  are  fitted  into  a framework  which  can  be  raised  or  depressed, 
so  as  to  bring  the  object  distinctly  into  focus. 

20.  Method  of  Measuring  the  Thickness  of  Cover-Glasses. — 
Thick  cover-glasses  are  of  no  use,  and  thin  ones — extra  thin,  so 
called — are  to  be  pre 
ferred.  A convenient 
thickness  is  0.16  mm. 

For  high  powers  this 
is  essential,  and  it  is 
well  to  measure  the 
thickness  of  the  cover- 
glasses,  and  to  reject 
all  those  above  a cer- 
tain thickness.  This 
is  conveniently  and 
rapidly  done  by  means 
of  the  instrument 
shown  in  fig.  26.  A 
clip  projecting  from  the  box  fixes  the  cover-glasses,  and  the  thick- 
ness is  given  by  an  indicator  moving  over  a divided  circle  on  the  lid 
of  the  box.  The  divisions  show  hundredths  of  a millimetre. 

21.  Camera  Obscura  Shade.  —When  one  has  to  continue  observing 
a microscopic  object  for  a long  time,  it  is  convenient  to  shade  the 
eyes  from  all  light  reaching  them,  except  that  transmitted  through 
the  eye-piece.  This  is  best  done  by  a blackened  screen  either  fixed 
to  the  microscope  or  arranged  in  front  of  the  microscope. ^ Some 
observers  place  the  microscope  in  a dark  chamber,  allowing  light  to 
fall  upon  the  mirror  through  an  aperture  in  its  front  wall.  Flogel^ 
has  designed  a camera  obscura  for  this  purpose. 

^ Scliiefferdecker,  Archiv  f.  wiss.  Mikr.,  p.  180,  1892. 

2 Zoolog.  AnzeigeVy  by  V.  Carus,  p.  566,  1883. 


Fig.  26.— Cover-glass  Tester. 


24 


PRACTICAL  HISTOLOGY. 


III.—NORMAL  OR  INDIFFERENT  FLUIDS. 


Not  unfrequently  living  tissues  or  fresh  tissue  elements  have  to 
be  examined  in  as  natural  a condition  as  possible,  and  obviously,  if 
a fluid  require  to  be  added  to  it,  the  fluid  must  be  of  such  a nature 
that  it  will  not  injuriously  affect  the  tissue  or  its  elements.  These 
fluids  are  spoken  of  as  indifferent  or  normal  fluids.  When  fresh, 
these  fluids  cause  very  slight  changes  in  the  tissues.  Amongst 
those  used  are — 

1.  Normal  Saline. — Dissolve  6 grams  of  pure  sodic  chloride  in 
looo  cc.  water. 

2.  Kronecker’s  Fluid. — It  consists  of 

Distilled  water  . . . . . lOO  cc. 

Sodic  chloride  . . . . . .6  gram. 

Soda  . . , . . . .o6  ,, 


3.  Blood  Serum.  —The  blood  is  allowed  to  clot,  and  the  serum, 
after  a day  or  so,  is  poured  away  from  the  clot.  In  a laboratory 
provided  with  a ‘‘centrifugal  apparatus*^  any  red  blood-corpuscles 
can  be  got  rid  of  by  “ centrifugalising  ” the  serum.  This  fluid 
does  not  keep  long,  and  must  be  fresh.  It  has  been  suggested  to 
add  a piece  of  camphor  to  it,  but  this  only  helps  to  preserve  it 
for  a short  time.  Iodine  is  sometimes  added  to  serum  to  form 
indised  serum,  but  this  is  by  no  means  an  indifferent  fluid 
(p-  25)- 

4.  Aqueous  Humour.  — With  a narrow  triangular  knife  puncture 
the  cornea  of  a freshly-excised  ox’s  eyeball  and  collect  the  aqueous 
humour  which  exudes.  If  only  a small  quantity  be  required,  it 
may  be  obtained  by  puncturing  the  excised  eye  of  a frog  with  a fine 
capillary  glass  pipette  (p.  4). 

5.  Fluid  of  Bipart  and  Petit : — 


Camphorated  water 
Distilled  water 
Glacial  acetic  acid 
Acetate  of  copper 
Chloride  of  copper 

It  is  very  useful  for  examining  animal  cells. 


75  cc. 

75  „ 

I n 

o.  30  gram. 

0.30  n 


IV.— DISSOCIATINO  FLUIDS. 

These  fluids  dissolve  or  soften  the  cement  substance  or  inter- 
stitial material,  e.g.,  of  epithelium,  connective  tissue,  and  thus 
facilitate  the  separation  of  the  histological  elements  from  each  other. 
In  some  cases,  in  isolating  tissue  elements,  it  is  well  not  to  have 


DISSOCIATING  FLUIDS. 


25 


too  much  fluid  in  proportion  to  the  morcel  of  tissue,  else  hardening 
is  more  apt  to  take  place. 

1.  Strong  Iodised  Serum.  — A strong  solution  should  be  kept, 
Avhiclt  may  be  diluted  as  required.  To  the  amniotic  fluid  of  a cow 
or  to  blood-serum  add  crystals  of  iodine,  and  keep  it  in  a stoppered 
bottle.  Shake  it  frequently.  At  first  very  little  iodine  is  dissolved, 
but  after  a time  (15-20  days)  the  solution  becomes  much  stronger, 
?.c.,  it  becomes  of  a deep-brown  tint. 

For  dissociating  a tissue,  a weak  iodised  serum  is  used.  A 
little  of  the  strong  fluid  is  added  to  fresh  serum  until  the  latter  has 
a light-brown  colour.  If  an  object  be  placed  in  the  dilute  iodised 
scrum  and  the  brown  colour  fades,  more  of  the  strong  solution 
must  be  added.  In  using  this  fluid,  take  a very  small  piece  of 
tissue,  the  size  of  half  a pea  or  less,  and  place  it  in  5 cc.  of  the 
fluid  in  a glass-stoppered  bottle.  After  a day  or  two  it  may  be 
dissociated  with  needles,  but  the  brown  tint  must  be  maintained  ; 
more  strong  fluid  must  be  added  if  putrefaction  is  to  be  pre- 
vented. 

2.  Dilute  Alcohol  (KanvieFs  Alcool  au  — This  fluid, 

devised  by  Ranvier,  and  sometimes  called  “one-third  alcohol,’^  is 
of  the  greatest  possible  service,  and  is  one  of  the  best  dissociating 
fluids  we  possess.  Mix  i part  of  96  per  cent,  alcohol  with  2 parts 
of  distilled  water.  It  dissociates  epithelial  and  other  tissues  in 
24-36  hours.  Use  a small  quantity  of  the  fluid  in  proportion  to 
the  tissue. 

3.  Chromic  Acid. — One  gram  in  1000  of  water.  This  requires 
two  days  to  a week,  according  to  the  tissue  placed  in  it. 

4.  Potassic  Bichromate. — Two  parts  in  1000  of  water,  f.c.,  .2 
per  cent.  It  is  very  useful  for  dissociating  epithelium  and  the 
nerve-cells  of  the  spinal  cord.  It  does  so  in  2-3  days. 

5.  Ammonium  Chromate  (5  per  cent.). — It  is  used  for  disso- 
ciating the  “rodded”  cells  of  the  renal  tubules,  cells  of  salivary 
glands,  Purkinje’s  fibres  of  the  heart,  &c.  It  acts  in  24-36  hours, 
and  the  chromate  must  be  well  washed  out  of  the  tissues  if  they 
are  to  be  preserved. 

6.  Caustic  Potash. — Thirty  to  35  parts  in  100  of  water.  It  acts 
ill  20-30  minutes,  rapidly  destroying  connective  tissue.  Water  must 
not  be  added  to  the  dissociated  tissue,  else  the  tissues  are  rapidly 
dissolved.  It  is  used  for  isolating  the  fibres  of  smooth  muscle,  or 
heart-fibres.  Examine  the  dissociated  tissues  in  the  dissociating 
fluid.  As  a rule,  tissues  so  dissociated  cannot  be  preserved,  but 
there  are  certain  exceptions. 

7.  Dilute  Osmic  Acid  (.1  per  cent.). — It  acts  in  24-48  hours 
according  to  circumstances,  and  is  well  suited  for  cells  containing 
fat  globules. 

4 


26 


PRACTICAL  HISTOLOGY. 


8.  Landois’  Fluid : — 


Neutral  ammouium  chromate  . . .5  grms. 

Potassium  phosphate 

Sodium  sulphate  . . . . ,, 

Distilled  water  . . . . . 100  ce. 

This  is  specially  useful  for  the  central  nervous  system.  Small 
pieces  must  lie  in  it  for  1-5  days.  (See  Lesson  on  N’ervous  System.) 

9.  Methyl  Mixture  or  SchiefFerdecker’s  Fluid : — 


Methylic  alcohol . 
Glycerine 
Distilled  water  . 


5 cc. 

so  „ , 

100  ,, 


It  is  better  to  prepare  it  fresh,  but  it  can  be  preserved  in  a 
glass-stoppered  bottle.  The  methylic  alcohol  rapidly 
evaporates.  The  tissues  remain  in  it  for  several  days, 
and  it  is  specially  useful  for  the  retina  and  central 
nervous  system.  I find  that  it  isolates  epithelial  cells 
in  one  to  two  days.  It  acts  very  much  like  Ranvier’s 
alcohol. 

10.  Other  fluids  are  referred  to  in  the  text,  e,g.^ 
baryta  water,  and  10  per  cent,  sodic  chloride  for 
tendon.  This  dissolves  the  cement  substance  of  epi- 
thelial cells  and  the  mucinoid  cement  of  connective 
tissue. 

11.  Digestion  Methods,  both  gastric  and  tryptic. 
Glass  arc  used  (see  text). 

The  special  uses  of  the  above-named  fluids  are 
referred  to  in  text  under  the  tissues  or  organs,  for 
which  each  is  specially  adapted. 

General  Directions  for  Dissociating  Tissues. — 
Always  use  a very  small  ])iece  of  the  tissue  or  organ,  and  place  it 
in  a not  too  large  quantity  of  the  dissociating  fluid.  Small  glass 
thimbles  (flg.  27)  are  very  useful  for  this  purpose. 


Fig.  27.' 

Tube  or 
Thimble  for 
dissociating 
small  pieces 
of  a tissue. 


V.— HOW  TO  TEASE  A TISSUE. 

To  separate  by  means  of  needles  the  elementary  parts  of  a tissue 
is  by  no  means  an  easy  task.  The  tissue  must  be  seen  distinctly, 
and  the  needles  must  be  so  used  as  not  to  break  up  the  parts,  but 
only  to  separate  them.  The  process  may  be  done  by  the  unaided 
eye  or  with  the  aid  of  a lens  or  dissecting  microscope  (figs.  24,  25). 
The  light  must  be  good,  and  an  appropriate  background  for  the 
object  should  be  selected.  If  the  tissue  is  colourless,  use  a black 


HOW  TO  TEASE  A TISSUE. 


27 


background,  e.g,^  black  paper;  if  coloured,  a white  one.  In  the 
latter  case,  however,  the  shadows  interfere  with  exact  vision,  and  it 
is  better  to  support  the  slide  upon  an  object  raised  slightly  above 
the  white  background.  This  is  readily  accomplished  by  placing  it 
over  a white  porcelain  capsule,  or  on  a photophore  (fig.  28),  into 
which  is  slipped  a piece  of  white  paper. 

In  a laboratory,  one  of  the  most  convenient  ways  is  to  have  the 
tables  painted  of  a black  or  very  dark  green  colour,  but  the  painting 
must  be  flat,  with  no  shining  varnish.  At  the  edge  of  the  table  is 
painted  a white  strip  i|  inches  broad.  Some  prefer,  to  burn  into 
the  surface  of  the  table  solid  paraffin  blackened  by  means  of  lam]i- 
black.  Others  use  porcelain  slabs  one-half  black  and  the  other 
white. 

Photophore  (fig.  28). — This  is  a small  wooden  box,  5 cm.  high, 
9 cm.  long,  and  9 cm.  broad.  The  upper  part  is  formed  of  glass. 
The  front  wall  of  the  box  is  wanting. 

Placed  obliquely  within  the  box  is  a 
mirror,  which  reflects  the  light  upwards 
through  the  glass  cover  and  the  slide  to 
the  eye  of  the  observer. 

In  dissociating  with  needles,  we  must  yiq.  28.— Photophore. 

have  some  knowledge  of  the  arrange- 
ment of  the  parts  of  the  object  to  be  teased,  such  as  the  direction 
of  the  fibres,  &c.  Take  a small  piece  only.  Always  tease  one  end 
of  the  tissue,  and  fix  the  latter  with  one  needle  while  the  parts  are 
separated  with  the  other  needle. 

One  of  the  most  convenient  combinations  is  that  of  Eternod, 
which  combines  a photophore  with  a turntable.  The  wheel  of  the 
latter  can  be  removed,  and  the  block  forms  not  only  a photophore, 
but  also  a surface  on  which  tissues  of  different  colours  can  be  teased 
and  mounted. 


VI— FIXING  AND  HARDENING  REAGENTS. 

Most  of  the  tissues  and  organs  must  be  hardened  in  suitable 
fluids  before  they  can  be  cut  into  sections.  A large  number  of 
fluids  of  various  kinds  are  used,  each  organ  or  tissue  requiring  its 
own  appropriate  fluid.  Some  organs,  e.g.,  bone,  are  too  hard  to  be 
cut  in  their  natural  cojidition ; they  must  be  decalcified  by  appro- 
priate fluids.  Amongst  others,  the  following  fluids  are  required, 
but  others  are  referred  to  in  the  text. 

A.  Alcohol. 

Alcohol  is  one  of  the  most  inij^ortant  hardening  fluids  used 


28 


PRACTICAL  HISTOLOGY. 


either  by  itself,  or  to  complete  the  hardening  processes  begun 
with  other  fluids.  Moreover,  it  is  almost  universally  used  for 
the  preservation  of  tissues  already  hardened.  There  are  three  kinds 
of  alcohol  used  for  histological  purposes,  viz.,  absolute  alcohol,  recti- 
fied spirit,  and  methylated  spirit. 

(a.)  Absolute  Alcohol  is  alcohol  without  water,  but  that  sold 
usually  contains  96  per  cent,  of  pure  alcohol,  and  is  sufficiently 
strong  for  microscopical  purposes.  If  an  absolutely  water-free 
alcohol  is  desired,  place  well-dried  potassic  carbonate  in  the  alcohol. 
This  rapidly  absorbs  the  moisture.  Or  powdered  and  heated  cupric 
sulphate — a white  powder — is  added.  If  water  is  present,  it  absorbs 
it,  and  becomes  blue  again.  Lowne  uses  slips  of  gelatine. 

(b.)  Rectified  Spirit  contains  84  per  cent,  of  spirit  and  16  per 
cent,  of  water. 

(c.)  Methylated  Spirit  contains  a little  wood  naphtha,  and  is 
nearly  as  strong  as  the  ordinary  absolute  alcohol,  and  may  be  made 
stronger  by  placing  some  well-dried  carbonate  of  potash  in  it.  The 
carbonate  of  potash  absorbs  any  water  present  in  the  alcohol.  Others 
use  cupric  sulphate  well  heated  in  a metallic  capsule,  until  it 
becomes  a white  powder.  When  it  becomes  blue  in  the  spirit  it 
must  be  replaced  by  new  CUSO4.  It  can  be  heated  and  used  again. 

Alcohol  is  used  of  various  strengths  for  hardening  purposes. 

Seventy-five  per  Cent.  Alcohol. — To  every  75  cc.  of  absolute 
alcohol  add  25  cc.  of  distilled  water. 

Seventy  per  Cent.  Alcohol. — To  every  70  cc.  of  absolute  alcohol 
add  30  cc.  of  distilled  water. 

Fifty  per  Cent.  Alcohol. — Take  equal  volumes  of  absolute 
alcohol  and  distilled  water. 

To  Harden  in  Alcohol  Alone. — The  tissues  are  placed  for  12-24 
hours  in  the  weaker  (50  per  cent.)  alcohol,  and  passed  through  the 
stronger  alcohols  (in  each  a day  or  thereby),  and  finally  kept  in  95 
per  cent,  alcohol  until  they  are  required. 

For  certain  special  purposes  the  tissues,  e,g.,  glands  and  struc- 
tures for  the  preservation  of  mitotic  figures,  are  ‘‘  fixed,’’  and  at  the 
same  time  hardened  by  being  placed  at  once,  and  while  as  fresh  as 
possible,  in  absolute  or  96  per  cent,  alcohol. 

B.  Chromium  and  its  Compounds. 

1.  Chromic  Acid  (Stock  Solution). — It  is  well  to  prepare  a 
strong  solution — 10  per  cent. — and  to  keep  this  as  a stock  to  be 
diluted  when  required.  Dissolve  10  grams  of  fresh  chromic  acid 
in  90  cc.  of  distilled  water. 

2.  Half  per  Cent.  Solution  of  Chromic  Acid. — To  50  cc.  of  the 
10  per  cent,  solution  add  950  cc.  of  distilled  water,  or  dissolve  i 
gram  of  chromic  acid  in  200  cc.  of  water.  Similar  solutions 


FIXING  AND  HARDENING  REAGENTS. 


29 


containing  .3  and  .2  per  cent,  chromic  acid  are  frequently  used,  and 
can  readily  be  made  from  the  stock-hottle. 

3.  Chromic  Acid  and  Spirit,  or  Klein’s  Fluid. — ?dix  2 parts  of 
chromic  acid  (.6  per  cent,  /.e.,  6 grams  in  1000  cc.)  Avith  i part  of 
methylated  spirit.  This  should  be  made  fresh,  and  kept  from  the 
light.  If  the  fluid  be  changed  often  it  hardens  tissues  in  8-10 
days. 

4.  Chromic  Acid  and  Bichromate  Solution. — Dissolve  i gram 
of  chromic  acid  and  2 grams  of  potassic  bichromate  in  1500  cc.  of 
water. 

5.  Muller’s  Fluid. — Dissolve  25  grams  of  potassium  bichromate 
and  10  grams  of  sodium  sulphate  in  1000  cc.  of  Avater.  Solution 
takes  place  slowly  at  the  ordinary  temperature.  Pound  the 
ingredients  in  a mortar,  add  the  Avater,  and  A\mrm  until  they  are 
dissolved.  It  takes  five  to  seven  Aveeks  to  harden  a tissue, 
according  to  the  size  of  the  tissue  placed  in  it.  This  fluid  is  A^ery 
extensively  used,  as  it  penetrates  into  the  tissues  and  hardens  them 
equally  throughout.  To  prevent  the  formation  of  fungi,  place  a 
piece  of  camphor  in  the  solution.  Muller’s  fluid  preserves  blood- 
corpuscles  in  their  original  form,  and  they  retain  their  yelloAv 
colour.  It  also  shrivels  very  slightly  the  tissue  elements. 

G.  Muller’s  Fluid  and  Spirit. — IMuller’s  fluid  3 parts,  and 
methylated  spirit  i part.  Mix,  and  alloAV  the  mixture  to  cool 
before  using  it.  When  tissues  are  hardened  in  it  they  should  be 
kept  in  the  dark. 

7.  Erlicki’s  Fluid.  — Dissolve  2.5  grams  of  potassium  bichromate 
and  .5  gram  cupric  sulphate  in  100  cc.  Avater.  It  should  be 
prepared  fresh.  It  hardens  more  quickly  than  Muller’s  fluid,  and 
after  the  first  day  or  tAvo  its  action  is  greatly  facilitated  by  keeping 
the  tissues  in  it  at  40°  C.  Ten  days  or  so  Avill  suffice  for  hardening 
under  these  conditions.  Experience,  hoAvever,  has  shoAvn  that  the 
process  of  rapidly  hardening  tissues  at  a comparatively  high 
temperature  is  not  so  satisfactory  as  that  conducted  at  a lower 
temperature. 

8.  Potassium  Bichromate. — Make  a 2 per  cent,  solution  by 
dissolving  10  grams  of  the  salt  in  500  cc.  of  Avater.  It  takes 
from  three  to  seven  Aveeks  to  harden  tissues,  and  is  one  of  the  best 
hardening  fluids  for  the  central  nervous  system. 

9.  Ammonium  Bichromate  is  used  in  the  same  Avay  and  of  the 
same  strength.  It  takes  much  longer  to  harden  than  8.  (See 
Central  Kervous  System.) 

10.  Ammonium  Chromate  (5  per  cent,  solution).  ^ — This  is  used 
for  hardening  the  kidney  and  other  secretory  glands,  the  mesentery 
of  the  neAvt,  (fee.  (see  text). 

Precautions  in  Connection  with  Chromium  Compounds. — Solu- 


30  > 


PPvACTICAL  HISTOLOGY. 


tions  of  pure  chromic  acid  do  not  penetrate  well  into  tissues, 
therefore  the  pieces  of  tissue  must  he  small.  In  the  case  of  tissues 
placed  in  MuHer'’s  fluid  or  i^otassic  bichromate,  they  are  hardened 
very  slowly.  In  the  case  of  most  organs  3-4  weeks  suffice,  hut  in 
the  case  of  the  brain  and  spinal  cord  it  takes  several  months  to 
harden  tissues  and  organs  in  these  fluids.  The  fluid  must  he 
frequently  changed,  and  it  must  be  large  in  amount.  The  forma- 
tion of  fungi  on  its  surface  may  be  prevented  by  adding  a piece  of 
camphor,  thymol,  or  naphthaline  to  the  fluid.  The  hardening 
process  is  accelerated  when  the  fluid  and  tissue  are  kept  at  3o°-4o° 
C.,  but  the  result  is  not  so  satisfactory  as  by  the  slower  cooler 
process. 

Except  for  special  purposes,  e.g.,  Weigert’s  method  for  the  cen- 
tral nervous  system,  the  chromic  acid  salts  must  be  thoroughly 
washed  out  of  the  hardened  tissue  or  organ.  This  is  done  by 
leaving  them  for  many  hours  or  days  in  a stream  of  running  water. 
In  all  cases  it  is  well  to  keep  the  fluids  and  tissues  in  a cool  place, 
and  in  some  cases  in  the  darh,  e.g.,  3 and  6. 

In  connection  with  the  hardening  of  tissues,  especially  those  of 
the  central  nervous  system,  it  is,  with  right,  insisted  upon  that  the 
hardening  fluid  should  he  frequently  changed.  I find,  however, 
and  my  observations  are  borne  out  by  other  observers,  that  after 
the  first  change  of  fluid  a very  satisfactory  result  is  obtained  by 
placing  the  organ  to  be  hardened  in  a very  large  volume  of  the 
hardening  fluid,  e,g.,  the  spinal  cord  of  a cat  or  dog  in  a litre  of  the 
hardening  reagent,  and  leaving  it  under  proper  conditions  until  the 
cord  is  hardened.  Stir  the  fluid  from  time  to  time. 

Chromic  acid  seems  to  form  a compound  with  the  tissues,  so 
that  it  is  not  easily  removed  from  them.  Tissues  hardened  in 
chromic  acid  are  not  readily  stained  by  carmine,  so  that  for  this 
purpose  it  is  better  to  use  hsematoxylin  or  safranin. 

C.  Acids  and  Acid  Mixtures. 

1.  Picric  Acid. — Make  a cold  saturated  solution  of  picric  acid  in 
water.  There  should  always  be  a large  excess  of  crystals  on  the 
bottom  of  the  bottle.  Place  a small  piece  of  tissue  in  the  solution 
for  6-24  hours.  The  tissues  are  to  be  afterwards  washed  in  70  per 
cent,  alcohol — not  water — and  transferred  to  95  per  cent,  alcohol. 

2.  Kleinenberg’s  Picro-Sulphuric  Acid. — To  100  cc.  of  a cold 
saturated  watery  solution  of  picric  acid  add  2 cc.  of  concentrated 
sulphuric  acid.  This  causes  a copious  precipitate.  After  twenty 
hours  filter,  and  to  the  filtrate  add  300  cc.  of  distilled  water. 

Tissues  are  placed  in  this  fluid  for  a comparatively  short  space  of 
time — from  a few  minutes  to  two  or  six  hours.  The  time  should 
never  exceed  six  hours.  The  liquid  must  be  changed  if  it  becomes 


FIXING  and  hardening  REAGENTS.  3 1 

turbid.  The  hardening  process  is  completed  in  alcohol.  It  is  well 
adapted  for  embryological  work. 

3.  Picro-Nitric  Acid  (P.  Mayer). — Some  substitute  nitric  acid  for 
the  sulphuric  acid,  but  for  our  purposes  there  is  no  advantage  in 
this.  Small  pieces  of  tissue  not  over  J cm.  in  diameter — prefer- 
ably those  that  contain  little  connective  tissue — are  hardened  for  1-3 
hours.  They  are  washed  with  alcohol  until  most  of  the  yellow 
colour  disappears.  Sections  can  be  stained  with  haematoxylin. 

4.  Nitric  Acid  {Altmann). — Use  a 3 per  cent,  watery  solution  of 
pure  nitric  acid.  This  has  a sp.  gr.  of  1.02.  Use  as  small  pieces 
as  possible,  and  leave  them  in  the  mix^ture  just  until  they  are  fixed, 
^.6.,  from  a quarter  to  half  an  hour.  Strong  solutions,  if  they  act 
too  long,  dissolve  the  chromatin.  The  tissues  are  then  hardened 
successively  in  70,  80,  and  90  per  cent,  alcohol.  It  is  particularly 
useful  for  preserving  the  nuclei  of  cells,  mitotic  figures,  embryological 
tissues,  and  the  retina. 

5.  Per^nyi’s  Fluid  : — 

Nitric  Hcid  (10  per  cent.)  . . .40  cc. 

Chromic  acid  (0.5  per  cent.)  . . . 30  ,, 

Alcohol  . . . . . . 30  ,, 

It  is  a light  greenish-blue  liquid,  and  is  specially  useful  for 
hardening  ova  and  embryos.  Time,  4-6  hours  for  a small  embryo 
and  4-12  hours  for  the  tissues  of  vertebrates.  The  tissue  is  trans- 
ferred direct  to  75  per  cent,  alcohol  without  previous  washing  in 
water.  Borax  carmine  may  be  added  to  it,  and  then  this  mixture 
hardens  and  stains  at  the  same  time  [Garhini). 

6.  Chromo-Formic  Acid  (Pabl’s  Fluid). — To  200  cc.  of  \ per 
cent,  chromic  acid  add  four  to  five  drops  formic  acid.  It  must  be 
freshly  prepared,  and  fresh  tissues — small  pieces — are  placed  in  it 
for  12-24  hours.  The  tissues  are  thoroughly  washed  in  water,  and 
hardened  in  alcohol  of  gradually  increasing  strength.  Sections  can 
be  stained  in  haematoxylin  and  safranin.  It  is  specially  useful  for  the 
study  of  mitosis  and  nuclei  generally.  It  has  this  advantage,  that 
tissues  hardened  in  it  do  not  afterwards  darken. 

7.  Chromo- Acetic  Acid  (^Flemming). — As  chromic  acid  by  itself 
is  apt  to  cause  shrinking  of  some  of  the  more  delicate  textures,  it 
has  been  proposed  to  mix  it  with  a substance  which  has  an  opposite 
elTect,  such  as  acetic  acid.  The  following  combination  is  specially 
recommended  by  Flemming  for  fixing  the  achromatic  spindle  in 
cells  : — 

Chromic  acid  ....  gram. 

Glacial  acetic  acid  . . . • A)  cc. 

Water  .....  100  ,, 

The  tissues  must  be  small  in  size,  not  above  3-5  mm.  in  diameter. 


32 


PRACTICAL  HISTOLOGY. 


They  are  hardened  for  twenty-four  hours  in  this  mixture,  and  after- 
wards washed  for  twenty-four  hours  in  water,  and  the  hardening 
completed  successively  in  70,  80,  and  90  per  cent,  alcohol — each  for 
12-24  hours.  Sections  can  be  stained  with  hsematoxylin. 

8.  Osmic  Acid. — Make  a i per  cent,  solution  by  dissolving  i 
gram  in  100  cc.  of  distilled  water.  This  substance  is  rather  ex- 
pensive, and  is  sold  in  sealed  glass  tubes.  Carefully  clean  the 
surface  of  the  tube,  snip  off  one  end  of  it,  and  place  it  with  the 
requisite  quantity  of  water  in  a glass-stoppered  bottle,  which  has 
been  carefully  cleaned,  so  that  it  does  not  contain  a trace  of 
organic  matter.  Organic  matter  decomposes  it  very  rapidly.  It  takes 
several  hours  to  dissolve.  Some  prefer  to  use  normal  saline  solution 
instead  of  water  to  dissolve  it.  Its  vapour  is  very  irritating  to  the 
eyes  and  mucous  membranes  generally.  It  should  be  preserved  in 
yellow-coloured  bottles  and  kept  in  the  dark.  If  a yellow  bottle  is 
not  available,  cover  a bottle  with  brown  or  black  paper. 

9.  Osmic  Acid  Vapour. — This  is  most  useful  for  thin  membranes 
and  glands.  Tissues  stain  readily  after  its  use. 

10.  Chromo-Aceto-Osmic  Acid  (Flemming’s  Fluid). — To  45  cc. 
of  I per  cent,  chromic  acid  add  12  cc.  of  2 per  cent,  osmic  acid, 
and  then  3 cc.  of  glacial  acetic  acid.  This  can  be  kept  for  a long 
time,  and  need  not  be  kept  in  the  dark.  It  is  specially  useful  for 
fixing  the  figures  in  cell-division  or  mitosis,  and  for  many  other 
purposes.  It  “fixes”  tissues  in  from  a few  hours  to  twenty-four 
hours  or  longer ; but  the  pieces  must  be  small,  2-3  mm.  thick,  as  it 
does  not  penetrate  deeply.  This  is  Flemming’s  “ strong  formula.” 

A weaker  fluid  is  sometimes  used,  and  is  prepared  as  follows  : — 


Osmic  acid  (i  per  cent.) 

Glacial  acetic  acid  (i  per  cent.)  . 
Chromic  acid  (i  per  cent.) 

Water  . , . . 


10  c.c. 
10  „ 
25  M 
55  M 


They  must  be  thoroughly  washed  by  being  kept  in  running 
water  for  twelve  hours,  and  then  hardened  in  the  various  strengths 
of  alcohol,  70,  80,  and  90  per  cent.,  each  for  twenty-four  hours. 
Sections  should  be  stained  as  soon  as  possible  after  they  are  made, 
as  on  keeping  they  do  not  stain  so  well.  They  may  be  stained  with 
safranin,  haematoxylin,  or  methyl-violet,  but  safranin  is  the  best 
(p.  75),  it  stains  the  chromatin  of  the  cells  a bright  red.  Tissues 
hardened  in  it  are  not  well  adapted  for  teasing. 

11.  Fol’s  Solution. — This  is  a modification  of  10.  It  contains 
less  osmic  acid,  and  is  used  more  generally.  To  2 cc.  of  1 per  cent, 
osmic  acid  add  25  cc.  of  i per  cent,  chromic  acid,  five  parts  of  2 per 
cent,  glacial  acetic  acid,  and  68  cc.  water. 

Precautions  with  Osmic  Acid  or  Solutions  containing  it. — If  a 


FIXING  AND  HARDENING  REAGENTS. 


33 


tissue  is  to  be  hardened  in  a certain  fluid,  and  to  be  treated  with 
osmic  acid  afterwards,  that  tissue  liad  better  not  be  put  into  alcohol 
if  it  contains  fat  or  fatty  particles,  as  the  alcohol  dissolves  the  fat. 
Thus,  a tissue  hardened  in  Muller’s  fluid  may  be  put  into  osmic  acid, 
and  the  fat-cells  will  still  be  blackened. 

It  has  recently  been  shown  that  the  prolonged  action  of  turpen- 
tine, toluol,  xylol,  ether,  and  creasote,  but  not  clove-oil  or  chloroform, 
will  decolorise  particles  of  oil  (fat)  blackened  by  osmic  acid.  This 
is  most  important  in  connection  with  the  osmic  method  for  studying 
the  absorption  of  fat  in  the  small  intestine  (Lesson  XXY.). 

Tissues  containing  fat  may  therefore  be  embedded  in  paraffin  after 
being  passed  through  chloroform  (p.  43). 

Osmic  acid  fixes  fresh  tissues  very  rapidly,  but  it  does  not  pene- 
trate deeply ; therefore  the  tissues  must  be  cut  into  very  small  pieces 
to  get  them  fixed  throughout  by  the  acid.  It  has  the  power  of 
differentiating  tissues,  the  nuclei  become  yellow,  fat,  and  the  nervous 
system  black.  After  twenty-four  hours  or  so,  the  tissues  are 
thoroughly  washed  in  water  and  hardened  in  90  per  cent,  alcohol. 
To  avoid  the  blackening  which  is  apt  to  occur  in  tissues  still  con- 
taining a trace  of  osmic  acid,  it  has  been  proposed  to  treat  the 
tissues  with  a weak  solution  of  cyanide  of  potassium,  and  then  to 
harden  in  alcohol.  It  enters  into  the  composition  of  several 
hardening  fluids  (p.  32),  and  Hermann’s  fluid  (Lesson  XXXV.). 

D.  Other  Hardening  Fluids. 

1.  Bichloride  of  Mercury  or  Corrosive  Sublimate. — A saturated 
watery  solution  contains  about  5 per  cent,  of  the  salt ; but  it  is 
much  more  soluble  in  alcohol,  especially  alcohol  of  50  to  60  per 
cent.  Make  a saturated  watery  solution,  and  also  a saturated 
alcoholic  solution.  A saturated  solution  in  0.5  XaCl  solution  i.s 
also  used. 

A cold  saturated  solution  is  best  made  as  follows  : — Place  60 
grams  of  it  in  1000  cc.  of  water,  and  dissolve  with  the  aid  of  heat. 
Filter  the  warm  solution  and  allow  it  to  cool.  On  cooling,  long 
white  needle-shaped  crystals  of  the  sublimate  separate.  The 
supernatant  fluid  is  used,  and  the  tissues,  which  must  not  be  more 
than  ^ cm.  in  diameter,  remain  in  the  fluid  from  1-3  hours,  accord- 
ing to  their  size.  After  fixation  they  are  hardened  in  70,  80,  and 
90  per  cent,  alcohol.  Xo  metallic  instruments  are  to  be  used. 
Use  glass  or  wooden  instruments. 

This  is  a most  excellent  hardening  reagent,  and  it  hardens  tissues 
with  great  rapidity,  so  that  tissues  must  not  be  left  in  it  for  too 
long  a time.  For  small  pieces,  a quarter  of  an  hour  or  thereby  is 
6 C 


34 


PRACTICAL  HISTOLOGY. 


sufficient;  for  large  pieces,  one  to  two  hours.  Tlie  pieces  when 
fixed  become  whitish  throughout. 

For  glands  and  glandular  structures  generally,  a half-saturated 
alcoholic  solution  is  most  useful,  ^.e.,  to  50  cc.  of  a saturated 
alcoholic  solution  add  50  cc.  of  70  per  cent,  alcohol.  Vignal 
recommends  that  to  100  cc.  of  this  mixture  there  be  added  5 to 
6 drops  of  nitric  acid.  The  pieces  of  glands,  4 mm.  cubes,  are 
hardened  in  one  hour  or  so.  The  hardening  is  completed  in 
alcohol. 

F.B, — All  the  corrosive  sublimate  must  be  washed  out  of  the 
tissue — by  alcohol,  not  water — otherwise  the  sections  will  be 
dotted  with  small  black  specks  or  star-like  or  needle-shaped  crystals 
of  the  salt,  or  the  salt  may  be  removed  by  adding  a few  drops  of  an 
alcoholic  solution  of  iodine  to  the  alcohol  used  to  complete  the 
hardening,  or  before  complete  hardening  in  alcohol  it  may  be 
steeped  for  2-3  days  in  70  per  cent,  alcohol,  to  which  a few  drops 
of  tincture  of  iodine  is  added. 

The  action  of  the  salt  may  be  accelerated  by  placing  the  tissues 
in  the  fluid  heated  to  38^  C.  Sections  stain  readily  with  all  the 
usual  staining  reagents. 

N.B. — Do  not  use  metallic  instruments  to  transfer  the  tissues 
from  one  vessel  to  another.  Use  glass  or  wood  or  horn. 

2.  Other  Fluids  for  special  purposes  are  mentioned  in  the 
text. 


General  Directions  on  Hardening. 

The  tissues  should  be  taken  from  the  body  as  soon  as  possible 
after  death,  and  transferred  as  soon  as  possible  to  the  hardening 
fluid. 

Any  blood  adhering  to  the  parts  may  be  removed  by  washing 
them  in  normal  saline  solution. 

With  a sharp  razor  cut  the  tissues  in  the  same  plane  in  which 
they  are  afterwards  to  be  cut  for  sections.  The  tissue  must  be  cut 
into  small  pieces,  ^.e.,  4 to  f inch  cubes,  except  in  the  case  of 
tissues  to  be  hardened  in  MiilleFs  fluid.  They  will  harden  better 
if  they  are  small,  say  i cm.  square. 

The  best  way  is  to  suspend  the  tissues  in  the  upper  half  of  the 
fluid,  which  should  always  be  many  times  the  volunie-^15-20 — 
of  the  tissue.  If  it  is  inconvenient  to  suspend  them,  cover  the 
bottom  of  the  jar  or  wide-mouthed  bottle  in  which  they  are  placed 
with  an  old  washed  rag  or  blotting-paper  to  prevent  the  tissue  from 
resting  directly  on  the  glass. 

The  liquid  must  be  changed  within  the  first  twenty-four  hours, 
and  again  on  the  second  day,  then  on  the  fourth,  eighth,  and  twelfth 


FIXING  AND  HARDENING  REAGENTS. 


35 


day,  and  once  or  twice  afterwards,  and  on  each  occasion  alter  the 
position  of  the  tissues.  If  the  fluid  becomes  turbid,  change  it  at 
once  (p.  30).. 

Label  each  bottle  carefully,  and  place  it  in  a cool  place.  Keep 
all  chromic  acid  and  solutions  containing  it  or  its  salts  in  the 
dark. 

Tissues  hardened  in  alcohol  and  picric  acid  must  not  be  placed 
in  water,  but  directly  into  the  various  strengths  of  aloohol, 
beginning  with  50  per  cent,  and  rising  to  95  per  cent.  Wash 
out  by  means  of  alcohol  as  much  of  the  picric  acid  as 
possible. 

For  other  tissues,  hardened  in  chromium  salts  (p.  30),  the  excess 
of  these  salts  may  be  removed  from  them  by  washing  in  water  (for 
certain  special  purposes,  this  is  omitted),  and  they  are  then  transferred 
first  to  weak  spirit,  in  which  they  may  remain  a few  days,  and  then 
to  the  stronger  alcohols  (p.  28).  To  avoid  the  deposits  which  occur 
in  chromic  acid  preparations  when  they  are  placed  in  alcohol,  keep 
them  in  the  dark.  If  kept  in  the  dark,  as  Ilans  Yirchow  has 
shown,  there  is  no  deposit  formed  when  a tissue  hardened  in 
chromic  acid,  or  Muller’s  fluid  is  placed  in  spirit.  Kept  in  this 
way,  I find  that  the  alcohol  remains  quite  clear  and  no  deposit 
forms. 


Scheme  for  the  Hardening  of  Tissues  (Garbini). 
Fresh  Object. 


Picric  acid  or  Chromic  acid  or 

solution  con-  its  compounds, 

taining  it. 


Alcohol 

(50  p.o.). 


in  dark. 


Alcohol  (70  p.c.) 
in  dark. 


Sublimate. 


Osmic  acid. 


Running  water.  Distilled  water.  Running  water. 


Alcohol  (50  p.c.)  Alcohol  (50  p.c.)  Cyanide  of  Potassium. 


(iodated). 

Alcohol  (70  p.c.)  Alcohol  (50  p.c.). 
(iodated).  1 

I ( 


Alcohol  (70  p.c.). 


36 


PRACTICAL  HISTOLOGY. 


Garbini  divides  hardening  reagents  into  the  two  following  classes, 
according  as  the  tissues — after  hardening — stain  readily  or  do  not 
stain  readily  with  carmine  : — 


Tissues  stain  readily  after 
hardening  in 


^ Vapour  of  osmic  acid.  , 
Corrosive  sublimate. 

Picric  acid  and  solutions  contain- 
ing it. 

Nitric  acid. 

Silver  nitrate  (weak  solution). 

^ Alcohol. 


f Chromic  acid  and  its  compounds. 
Tissues  stain  with  diffi-  | Osmic  acid  in  solution. 

culty  after  hardening  ^ Silver  nitrate  (strong  solution), 
in  I Chloride  of  gold. 

t Perchloride  of  iron. 


Alcohol,  picric  acid  and  its  compounds,  and  nitric  acid,  coagulate 
the  albumen  of  the  tissues.  Chromic  acid,  or  solutions  containing 
it,  or  its  salts  form  chemical  compounds  with  substances  in  the 
tissues.  Osmic  acid,  mercuric  chloride,  and  gold  are  decomposed 
when  they  come  in  contact  with  the  tissues,  and  are  deposited  in 
the  tissues  as  an  inorganic  precipitate. 


VII.— DBCALCIPYINa  FLUIDS. 

General  Directions. — U se  small  parts  of  the  tissue  or  organ,  and 
a large  quantity  of  the  decalcifying  solution.  Kenew  the  latter 
often.  In  all  cases  harden  the  tissue  thoroughly,  e.f/.,  in  alcohol 
or  Muller’s  fluid,  before  it  is  decalcified.  After  decalcification  every 
trace  of  the  decalcifying  fluid  must  be  thoroughly  removed  by  pro- 
longed washing  (for  a day  or  two)  in  water  or  other  fluid.  The 
tissue  is  then  finally  hardened  in  alcohol. 

1.  Chromic  Acid. — . i to  .5  per  cent. 

2.  Chromic  and  Nitric  Acid  Fluid. 

Chromic  acid  . . . . .1  gram. 

Water  ......  200  cc. 

Nitric  acid  . . . . . 2 ,, 

3.  Chromic  Acid  and  Hydrochloric  Acid  Fluid  {BayerVs  Fluid). 

Chromic  acid  .....  i part. 

HCl  . . . . . . I „ 

Water  ......  100  parts. 

This  is  specially  good  for  young  bones  and  for  ossifying  bone, 
but  it  is  solely  for  decalcifying. 

4.  Saturated  Solution  of  Picric  Acid. — The  solution  must  be 


DECALCIFYING  FLUIDS. 


37 


saturated  and  kept  saturated.  This  is  done  by  keeping  some 
crystals  of  picric  acid  in  the  bottom  of  the  bottle.  There  must  be 
a large  volume  of  fluid,  and  the  bone  should  be  suspended  in  the 
fluid.  It  usually  requires  a fortnight  to  decalcify  a small  bone. 
Picric  acid  acts  as  a fixing,  hardening,  and  staining  reagent. 

After  the  bone  is  decalcified  it  should  be  washed  and  kept  in 
spirit — not  water — until  no  more  yellow  stain  is  given  up  to  the 
alcohol.  This  rule,  however,  is  not  rigidly  followed. 

5.  V.  Ebner’s  Fluid. — This  fluid  prevents  the  ground  substance 
of  Ijone  from  swelling  up.  Sections  should  be  examined  in  lo  per 
cent,  solution  of  sodic  chloride,  if  it  be  desired  to  see  the  fibrillar 
structure  of  bone,  but  sections  of  bone  softened  in  this  way  may  be 
mounted  in  other  media,  if  it  be  desired  to  see  the  other  details 
in  the  structure  of  bone.  (See  Bone.)  It  has  the  following  com- 
position : — 


Alcohol 
W ater  . 

Sodic  chloride  . 
Hydrochloric  acid 


500  cc. 

100  ,, 

2.5  grams. 
2.5  cc. 


6.  Picro-Sulphuric  Acid  or  Picro-Nitric  Acid  (p.  31). 

7.  Arsenic  Acid. — I find  that  a 4 per  cent,  solution  of  this  acid 
rapidly  decalcifies  a bone  at  30°  to  40°  C.  The  tissues,  after  harden- 
ing in  alcohol,  are  well  preserved  and  stain  readily. 

8.  Chromo-Osmic  Acid  {Haug). 

Osmicacid(i  percent.)  . . .10  cc. 

Chronuc  acid  (i  per  cent.)  . . . 25  ,, 

Water  . . . . . • 65  ,, 


Very  useful  for  delicate  tissues,  e.g.^  very  young  developing  teeth. 
Wash  in  water  and  harden  in  70  per  cent,  alcohol. 

9.  Phloroglucin  Method. — Dissolve  with  the  aid  of  gentle  heat 
I gram  of  phloroglucin  in  10  cc.  pure  non-fuming  nitric  acid.  To 
the  red  fluid  add  100  cc.  of  10  per  cent,  watery  solution  of  nitric 
acid.  The  following  mixture  acts  more  slowly  : — 


Phloroglucin 
Nitric  acid 
Alcohol 
Water  . 


I gram. 
5 cc. 

70  ,, 

30  .. 


Phloroglucin  itself  does  not  decalcify,  it  only  protects  the  tissues 
from  the  action  of  the  nitric  acid.  By  this  method  decalcification 
can  be  done  very  rapidly,  even  in  a few  hours.  I find  that  a portion 
of  a human  clavicle  about  an  inch  in  length  is  softened  in  12-16 
hours,  the  outer  surface  being  stained  slightly  red. 

Methods. — The  bone  should  be  cut  into  short  pieces  and  placed 


38 


PRACTICAL  HISTOLOGY. 


in  a large  volume  of  the  fluid.  If  chromic  acid  be  used,  the  bone 
must  be  first  hardened  in  this  fluid.  Place  it  in  .i  per  cent, 
chromic  acid  for  twenty-four  hours , renew  the  fluid,  but  use  .2  per 
cent.,  and  after  a week  use  .5  per  cent. ; shake  the  vessel  from  time 
to  time,  to  bring  new  fluid  into  contact  with  the  tissue. 

If  a more  rapid  process  is  desired,  after  the  bone  has  been  two  or 
three  days  in  dilute  chromic  acid  (.2  per  cent.),  use  the  chromic 
and  nitric  acid  fluid.  Decalcification  requires  about  fourteen  days. 

To  test  for  the  removal  of  all  the  salts,  push  a needle  into  the 
bone,  or  make  a section  with  a blunt  razor.  Obstruction  in  either 
case  denotes  that  the  bone  has  not  been  sufficiently  decalcified.  In 
most  cases,  the  bony  tissues  should  be  hardened  before  they  are 
decalcified.  This  is  specially  the  case  in  connection  with  bone 
softened  in  chromic  acid.  It  is  better  to  harden  them  first  in 
MiillePs  fluid,  and  then  to  decalcify  them  in  chromic  and  nitric 
acid  fluid  (p.  36). 

Bone  decalcified  in  chromic  acid  must  be  thoroughly  washed  in 
running  water  for  many  hours  to  remove  all  the  chromic  salts,  and 
is  then  transferred  to  70  per  cent,  spirit  and  kept  in  the  dar\  other- 
wise there  will  be  a copious  deposit.  Renew  the  spirit,  and  transfer 
tbe  tissue  to  strong  alcohol,  still  keeping  it  in  the  dark. 

If  a bone  is  to  be  softened  in  picric  acid,  it  may  be  placed  at  once 
in  this  fluid,  with  the  precaution  indicated  at  p.  37.  It  need  not 
be  kept  in  the  dark,  but  it  is  better  to  remove  as  much  as  possible 
of  the  yellow  stain  by  means  of  alcohol.  It  decalcifies  somewhat 
more  slowly  than  chromic  acid. 


VIII.— METHOD  OF  PREPARING  TISSUES  AND 
ORGANS  FOR  MICROSCOPICAL  EXAMINA- 
TION. 

As  most  of  the  tissues  require  to  be  hardened,  and  it  is  frequently 
impossible  to  obtain  human  tissues  sufficiently  fresh,  recourse  must 
be  had  to  the  fresh  tissues  of  animals.  As  frequently  as  possible, 
however,  human  tissues  should  be  secured.  Most  of  the  tissues 
may  be  obtained  from  a cat,  rabbit,  or  guinea-pig,  and  for  certain 
special  purposes  the  dog,  frog,  newt,  and  salamander  are  used. 

The  cat,  rabbit,  or  guinea-pig — or,  better,  all  three — are  killed 
by  chloroform.  The  animals  are  placed  in  an  air-tight  box — a 
large  saucepan  does  very  well — along  with  a “^sponge  saturated  with 
chloroform.  Small  animals  may  be  chloroformed  under  a bell-jar. 
As  soon  as  the  animal  is  dead,  open  the  thorax  by  a longitudinal 
incision  through  the  costal  cartilages — right  and  left — raise  the* 


METHOD  OF  PREPARING  TISSUES  AND  ORGANS.  39 


sternum,  expose  the  pericardium,  open  it,  and  with  a j)air  of  scissors 
make  a snip  into  the  right  auricle  of  the  heart,  and  allow  the  animal 
to  bleed  freely. 

It  is  best  to  begin  by  removing  the  brain  and  spinal  cord.  They 
are  hardened  in  Muller’s  fluid  or  potassic  bichromate  (2  per  cent.), 
and  must  be  placed  in  a large  volume  of  fluid.  A few  spinal 
ganglia  should  also  be  found  and  hardened  in  the  same  way. 

Remove  the  trachea  and  lungs,  and  fill  the  lungs  and  trachea  of 
the  rabbit  with  a ^ per  cent,  solution  of  chromic  acid. 

This  is  readily  effected  by  tying  a funnel  into  the  trachea  and 
pouring  in  the  fluid.  By  squeezing  the  lungs  gently  much  air  is 
forced  out,  and  the  fluid  gradually  runs  into  and  distends  the 
lungs,  which,  when  distended,  are  placed  in  a large  volume  of  the 
same  fluid.  The  chromic  acid  and  spirit  mixture  may  be  used 
instead  of  pure  chromic  acid. 

Fill  the  windpipe  and  lungs  of  the  guinea-pig  with  ^ per  cent, 
silver  nitrate.  (See  Lungs.) 

Remove  the  heart,  and  harden  it  in  alcohol,  after  washing  away 
any  blood  with  normal  saline. 

The  central  tendon  of  the  diaphragm  may  be  preserved  for 
silvering.  (Lesson  IV.) 

The  omentum  and  mesentery,  if  desired,  are  silvered.  (Lesson 

Open  the  abdomen,  remove  the  liver,  cut  it  into  small  pieces  ^ 
harden  some  pieces  in  Muller’s  fluid,  and  others  in  spirit. 

Take  out  the  tongue  and  oesophagus ; harden  them  in  Muller’s 
fluid. 

Open  the  stomach  and  intestine,  wash  away  any  food  residues 
by  means  of  normal  saline.  Harden  part  of  the  stomach — cardiac 
and  pyloric — in  absolute  alcohol,  other  pieces  in  Muller’s  fluid, 
and  others  in  corrosive  sublimate,  and  small  pieces  in  osmic  acid. 
(See  Lesson  on  Stomach  and  Intestine.) 

The  duodenum  and  small  and  large  intestine  are  hardened  in 
the  same  way,  although  the  bichromate  and  chromic  acid  mixture 
(p.  29)  is  particularly  good  for  the  small  intestine. 

The  salivary  glands  and  pancreas  are  removed  and  hardened  by 
the  methods  given  under  these  headings,  ^.6^,  small  pieces  are  placed 
in  each  of  the  following  solutions  : — Absolute  alcohol,  osmic  acid, 
corrosive  sublimate,  &c. 

Remove  the  lower  jaw,  cut  it  into  short  pieces,  place  it  in  .2  per 
cent,  chromic  acid  for  a few  days,  and  then  decalcify  it  in  chromic 
and  nitric  fluid.  This  will  yield  sections  of  softened  tooth. 

Remove  the  kidneys,  cut  one  longitudinally  and  the  other  trans- 
versely. Using  the  kidneys  of  different  animals,  harden  pieces  of 
each  in  the  following  fluids  : — Muller’s  fluid,  chromic  acid  and  spirit. 


40 


PRACTICAL  HISTOLOGY. 


ammonium  chromate,  and  corrosive  sublimate.  Other  methods  of 
preparing  the  kidney  are  referred  to.  (Lesson  on  Kidney.) 

The  bladder  is  best  hardened  in  chromic  acid  and  spirit  mixture, 
or  in  Muller’s  fluid. 

Harden  the  spleen,  without  cutting  into  it,  in  Muller’s  fluid. 

The  suprarenals  may  be  hardened  in  picro-sulphuric  acid. 
(Lesson  on  Suprarenal  Capsules.) 

Small  lymphatic  glands  from  the  region  of  the  neck  or  sub- 
maxillary  region  are  hardened  in  alcohol,  while  others  are  injected 
with  silver  nitrate  and  osmic  acid.  (Lesson  on  Lymphatics.) 

If  desired,  the  large  nerve-trunks  may  be  removed  and  hardened 
as  indicated  in  Lesson  on  Kerves,  or  the  smaller  branches  of  nerves 
may  be  used  for  showing  the  effects  of  the  action  of  certain  reagents 
on  nerve-fibres. 

Remove  some  of  the  long  bones,  leaving  in  each  case  the  peri- 
osteum attached  to  the  bone.  Cut  the  bones  into  pieces  about 
^ inch  long,  and  place  them  for  a week  in  ^ per  cent,  chromic  acid, 
and  then  decalcify  them  with  picric  acid,  or  chromic  and  nitric 
acid  fluid,  or  Ebner’s  fluid.  (Lesson  XIII.) 

In  every  case  decalcify  the  ends  of  the  bones,  so  as  to  have  a 
section  which  will  demonstrate  the  relation  between  the  articular 
cartilage  and  the  osseous  tissue. 

Place  small  pieces  of  striped  muscle  in  ^ per  cent,  chromic  acid, 
and  other  pieces  in  alcohol. 

Xerves,  with  the  precautions  given  in  Lesson  on  Xerves,  are 
hardened  in  osmic  acid,  potassic  bichromate  (2  per  cent.),  alcohol, 
or  picric  acid. 

Lor  the  methods  of  hardening  the  eye,  ear,  nose,  see  the  Lessons 
on  these  subjects. 

The  testis — very  small  pieces — is  best  hardened  in  Flemming’s 
mixture,  and  larger  pieces  in  Muller’s  fluid. 

For  the  methods  of  hardening  the  ovary.  Fallopian  tube,  and 
uterus,  see  the  Lessons  on  these  subjects. 

— Label  every  bottle,  and  write  on  the  bottle  the  name  of 
the  hardening  fluid  used,  and  the  dates  on  which  it  was  changed. 


I X.— EMBEDDING. 

This  is  necessary  for  many  tissues  ; the  piece  of  tissue  may  be 
either  too  small  to  be  cohvenienLly  held  in  the  hand,  or  its  parts 
may  tend  to  fall  asunder  before  or  after  they  are  cut. 

There  are  two  methods,  one  simple  embedding,  where  the  tissue 
is  simply  fixed  or  placed  in  another  medium  to  hold  it  while  it  is 


EMBEDDING. 


41 


being  cut,  and  the  other  interstitial  embedding,  where  the  sub- 
stance used  for  the  embedding  process  is  made  to  penetrate  into 
the  interstices  of  tlie  tissue. 

A.  Simple  Embedding. — 1.  The  tissue  may  be  clamped  between 
two  pieces  of  carrot,  scooped  out  to  receive  it,  or  in  elder  pith,  or 
(what  is  very  convenient)  between  two  pieces  of  amyloid  or  waxy 
liver  hardened  in  alcohol. 

2.  Paraffin.  — It  is  sometimes  desirable  to  surround  the  tissue 
with  paraffin  or  some  such  medium.  The  embedding  medium 
should  be  about  the  same  degree  of  hardness  as  the  tissue. 

Two  paraffins  are  required,  a hard  paraffin  melting  at  60°  and  a 
soft  one  at  45°  C.  For  use  they  may  be  mixed  as  follows  : — 


{Hard  paraffin 
Soft  paraffin 

{Hard  paraffin 
Soft  paraffin 


I part 
I ,, 


1 ,, 

2 parts. 


Two  parts  of  the  hard  paraffin  and  one  of  the  soft  yield  a mixture 
which  cuts  well  when  the  temperature  of  the  room  is  21°  C. 
(70°  F.),  but  a softer  paraffin  is  easily  made  by  mixing  two  parts  of 
the  hard  parafhn  with  one  part  of  chrisma  or  vaseline.  The 
mixture  can  be  made  softer  by  the  addition  of  a little  more 
vaseline,  and  harder  by  adding  more  paraffin.  The  paraffin 
mixture  is  heated  on  a water-bath  or  sand-bath  until  it  melts, 
but  its  temperature  is  raised  as  little  as  possible  above  its 
melting-point.  It  is  convenient  to  melt  it  in  a porcelain 
dish  with  a wooden  handle.  The  tissue  is  removed  from 
alcohol,  the  surplus  alcohol  removed  by  wiping  it  with  blotting- 
paper,  until  the  surface  is  dry.  It  is  then  placed  in  melted  paraffin, 
and  retained  in  it  until  the  paraffin  solidifies.  The  melted  paraffin 
can  be  run  into  embedding  boxes  of  paper  (fig.  30),  or  the  embed- 
ding L’s  may  be  used  (p.  44),  but  this  simple  method  is  now  but 
rarely  "ised.  It  has  been  almost  entirely  displaced  by  the  following 
method. 

B.  Infiltration  Method  or  Interstitial  Embedding. 

1.  Embedding  in  Gum. — The  tissues  after  being  hardened  must 
have  all  their  alcohol  removed  by  prolonged  soaking  in  water. 
They  are  then  transferred  to  gum  mucilage,  or  a mixture  of  gum  and 
syrup,  in  which  they  can  be  preserved  until  they  are  required  for 
freezing,  if  freezing  is  to  be  the  process  used  for  cutting  the  sections. 

Tissues  saturated  with  and  embedded  in  gum  mucilage  may  be 
hardened  in  alcohol  and  then  cut.  The  sections  are  placed  in 
water,  which  dissolves  out  the  gum. 

2.  Saturation  with,  or  Infiltration  with,  and  Embedding  in 
Paraffin — Interstitial  Embedding  -^In  this  case  the  embedding 


42 


PRACTICAL  HISTOLOGY. 


medium  is  made  to  penetrate  into  the  tissue,  and  when  it  sets,  it  thus 
supports  all  its  component  parts.  This  method  is  extremely  valuable, 
especially  for  brittle  and  friable  tissues,  and  is  largely  used.  More- 
over, the  tissues  once  embedded  can  be  kept  in  a box,  each  duly 
labelled,  for  any  length  of  time. 

Make  a mixture  of  two  parts  of  hard  paraffin  and  one  part  of 
soft ; place  the  mixture  in  a small  copper  pan  or  capsule  in  a hot- 
air oven,  kept  at  a constant  temperature  by  means  of  a gas  regu- 
lator. The  gas  supply  must  be  so  arranged  that  the  thermometer 


Fig.  29. — Mayer’s  Paraffin  Embeddij^  Batli,  as  made  by  Jung  of  Heidelberg. 

steadily  registers  at  most  1°  C.  above  the  melting-point  of  the 
paraffin.  Or  the  paraffin  may  be  melted  and  kept  melted  in  a 
little  copper  vessel,  placed  in  a hot- water  bath,  kept  at  a constant 
temperature,  as  shown  in  fig.  29.  The  temperature  is  kept  con- 
stant by  means  of  a gas  regulator,  E, ; Z is  for  filling  the*  instru- 
ment witli  water ; a,  7?,  c,  are  embedding  vessels  and  pots. 

The  tissues  to  be  saturated  and  embedded  should  not  be  large, 
and  they  must  be  thoroughly  dehydrated ; keep  them,  therefore, 
several  hours  in  absolute  alcohol.  Place  them  direct  into  tur- 
pentine, creosote,  benzol,  toluol,  or  xylol — some  use  chloroform,  but 


EMBEDDING. 


43 


turpentine,  toluol,  or  xylol  is  to  be  preferred.  Tlie  turpentine  or 
xylol  penetrates  into  the  tissue,  displaces  the  alcoliol,  and  makes 
the  tissue  itself  transparent.  If  the  tissue  be  very  small,  this  will 
be  done  in  an  hour  or  so ; if  it  be  larger,  of  course  a longer  time 
will  be  required.  Thus  the  time  may  vary,  according  to  the  size 
and  nature  of  the  tissue,  from  one  to  six  hours. 

Transfer  the  specimens  from  the  clarifying  medium,  and  place 
them  in  the  melted  paraffin,  where  they  may  remain  2-10  hours, 
according  to  the  size  and  nature  of  the  tissue.  By  tlui  end  of  that 
time  they  will  be  thoroughly  impregnated  or  saturated  with  the 
paraffin. 

For  delicate  tissues,  however,  it  is  better  not  to  transfer  them 
direct  from  the  clarifying  medium  to  pure  paraffin,  but  to  place 
them  first  of  all  in  a mixture  of  toluol  and  paraffin,  or  turpentine 
and  paraffin,  for  an  hour  or  two.  In  the  thermostat  at  S^°~55° 
the  toluol  gradually  evaporates,  so  that  nearly  pure  paraffin  remains, 
and  the  saturation  with  paraffin  has  been  accomplished  more 
gradually.  The  object  is  then  finally  }>laced  in  pure  paraffin  for 
several  hours. 

Some  prefer  chloroform  as  a clarifying  agent  and  as  a solvent  for 
paraffin  instead  of  toluol  or  turpentine.  Objects  transferred  from 
absolute  alcohol  at  first  float  on  the  surface  of  the  chloroform,  but 
as  the  latter  penetrates  them  and  displaces  the  alcohol  they  sink. 
They  are  then  embedded  in  a chloroform  paraffin  mixture  and 
finally  in  imre  paraffin. 

The  time  (in  liours)  required  for  immersion  in  the  several  fluids 
for  pieces  of  tissue  of  various  sizes  is  approximately  as  follows 
{Bolirti  and  Oppel) : — 


Small  Objects 

Objects 

Larger 

less  than 

about  5 mm. 

Objects  about 

Very  large  Objects. 

1 mm.  diam. 

diam. 

5 mm.  diam. 

Absolute  alcohol 

2 

6 

24 

( A longer  time, 
) but  at  the  ex- 

Toluol  or  turpentine 

1 

T 

2-3 

3-4 

1 pense  of  their 
( quality . 

Objects  are  now  placed  in  the  thermostat  or  bath. 

Toluol  or  turpen-  ) 

1 

A 

ft 

tine-paraffin  ( 

D 

Paraffin  . 

1 

2 

2 

3-4 

Objects  may  remain  longer  in  the  paraffin  without  damage, 
provided  they  have  been  completely  dehydrated. 


44 


PRACTICAL  HISTOLOGY. 


The  method  of  interstitial  embedding  is  particularly  useful  for 
tissues  stained  “ in  balk  or  en  inassa.  The  tissues  or  organ  may 
be  stained  before  the  process  is  begun,  or  sections  may  be  cut  and 
stained  afterwards. 

To  Stain  “in  bulk”  before  embedding. — Pieces  of  the  tissue  a 
few  millimetres  square  are  placed  in  borax-carmine  or  Kleinen- 
berg’s  logwood  for  10-24  4^  hours,  according  to  the  size  and 

nature  of  the  tissue.  If  they  be  placed  in  borax-carmine,  the  pieces 
are  transferred  to  acid  alcohol  for  24  hours,  and  then  transferred  to 
various  strengths  of  alcohol,  and  finally  to  absolute  alcohol,  by 
which  they  are  completely  dehydrated.  The  tissues  stained  with 
Kleinenberg’s  logwood  are  well  washed  in  spirit,  and  transferred 
to  absolute  alcohol  to  be  dehydrated.  These  dehydrated  stained 
masses  are  then  placed  in  turpentine  or  xylol,  and  then  in  melted 
paraffin,  as  described  above. 

Process  of  Embedding  in  Paraffin. — This  is  the  same  both  for 
the  simple  and  the  saturation  methods.  Use  embedding  L’s,  which 
consist  of  two  L-shaped  pieces  of  lead  about  ^ inch  high,  the  long 

arm  about  2 inches  long  and  the  short 

one  f inch.  Their  inner  surfaces  are 

moistened  with  glycerine,  and  the  Us 

themselves  are  placed  on  a piece  of  glass,  coated  with  glycerine,  to 
enable  the  paraffin  to  separate  easily,  with  the  long  limb  of  the 
one  in  contact  with  the  short  limb  of  the  other,  thus  making  a 
rectangular  box,  the  size  of  which  can  be  increased  as  required. 
Fill  the  trough  with  melted  paraffin. 

Take  the  tissue — if  for  simple  embedding — direct  from  alcohol ; 
dry  its  surfaces  with  bibulous  paper,  to  remove  any  alcohol  which 
would  prevent  the  paraffin  from  adhering  to  it;  pour  the  melted 
paraffin  into  the  trough,  transfix  the  tissue  with  a fine  pin,  plunge 
it  into  the  paraffin  just  when  the  latter  begins  to  set  at  the  edges, 
move  the  tissue  in  the  still  fluid  paraffin  to  one  end  of  the  trough, 
and  hold  it  there  until  the  mass  sets  around  it. 

If  the  tissue  has  been  previously  saturated  with  paraffin,  the 
trough  is  filled  as  before,  and  the  tissue,  saturated  with  paraffin,  is 
taken,  by  means  of  a hot  needle,  from  the  fluid  warm  paraffin,  and 
fixed  in  the  trough  in  the  same  way  as  described  above.  .It  is  not 
always  necessary  to  transfix  the  tissue  with  a pin  or  needle,  but  it 
is  sometimes  convenient  to  do  so.  Insert  the  needle  in  the 
direction  of  the  cutting  plane,  thus  indicating  afterwards  (when 
the  mass  is  set)  the  direction  in  which  the  section  is  to  be  cut.  A 
little  paraffin  may  be  poured  into  the  trough,  and  when  it  just 
begins  to  set,  the  tissue  is  laid  on  it,  and  another  layer  of  melted 
paraffin  is  poured  over  it  as  soon  as  its  surfaces  are  set.  Place  the 
whole  under  the  tap,  and  allow  cold  water  to  run  over  it  to 


EMBEDDING. 


45 


accelerate  the  cooling,  as  paraffin  cooled  rapidly  is  more  homogeneous 
and  cuts  better  than  Avhen  it  is  cooled  slowly.  ' In  about  half-an- 
hour  the  paraffin  has  set,  and  can  be  removed  from  the  mould. 

Embedding  Boxes  (fig.  30)  may  be  used.  These  are  readily 
made  from  a rectangular 

piece  of  writing-paper  b'  d'  d' 

folded  to  the  size  required. 

The  paper  is  first  folded  ^ | l> 

along  the  lines  ad  and  })h\  ” . 

then  along  cc'  and  dd\ 
always  folding  the  paper 
towards  the  same  side. 

The  diagonals  AA'-DD' 
are  indented  by  means  of 
the  point  of  a lead  pencil, 
or  the  paper  is  folded 

along  these  lines.  These  fio.  3o.-Embeddiui 

corners  are  then  bent  up 
between  the  fore-finger  and  thumb,  and  then  bent  round,  so  as  to 
be  applied  to  the  sides  AB  and  CD  of  the  oblong,  and  are  fixed 
there  by  turning  down  the  flaps  ,/f'. 

Embedding  in  Paraffin. — The  folio  wingseheme  shows  the  stages : — 


/ 

B 

D 

/ 

r 

A 

C 

d 

; Box. 


(1.)  Harden,  either  from  the  first  or  subsequent  to  other  agents 
in  absolute  alcohol. 

(2.)  Xylol  or  turpentine  (24  hours). 

(3.)  In  warm  paraffin  fluid  at  50°  C.  (1-12). 

(4,)  Embed  and  allow  paraffin  to  cool. 

(5.)  Cut  sections,  and  in  some  cases  fix  them  on  slide  with 
a fixative  (p.  60). 

(6.)  Remove  paraffin  by  turpentine  or  xylol. 

(7.)  Alcohol  to  remove  xylol. 

(8.)  Add  water  and  then  stain,  &c. 


3.  Embedding  in  Celloidin. — This  method  is  specially  valuable 
where  the  parts  of  an  organ  when  cut  into  sections  are  apt  to  fall 
asunder.  It  is  specially  valuable  in  such  as  those  of  the  ovary, 
central  nervous  system,  retina,  &c. 

Celloidin  is  a form  of  nitro-cellulose  or  pyroxylin,  or  solidified 
collodion,  and  is  sold  in  two  forms,  one  in  tablets  and  the  other  in 
cuttings.  E.  Schering’s  is  the  best,  and  the  form  sold  in  “ cuttings  ” 
is  to  be  preferred.  Do  not  let  its  solution  dry,  as  it  is  then  difficult 
to  redissolve  it. 

This  method  was  invented  by  Duval,  who  used  collodion,  and 
improved  by  Schietferdecker.  Prepare  the  solutions  of  celloidin 
by  dissolving  the  latter  in  equal  parts  of  absolute  alcohol  aiiu 


46 


PRACTICAL  HISTOLOGY. 


ether.  The  first,  or  weaker  solution,  is  made  of  a thin  consistence 
like  collodion  duplex,  the  other  is  made  stronger,  until  it  has  a thick 
syrupy  consistence. 

The  hardened  tissue  is  placed  for  some  time  in  absolute  alcohol, 
and  then  for  several  days,  or  until  it  is  completely  saturated,  in  a 
mixture  of  equal  parts  of  absolute  alcohol  and  ether.  After  this  it 
is  placed  in  a glass-stoppered  bottle  in  the  thin  solution  of  celloidin 
until  it  is  completely  saturated  with  it  (2-3  days).  Transfer  the 
tissue  to  syrupy  celloidin,  and  let  it  remain  there  for  several  days 
until  the  tissue  is  thoroughly  infiltrated  with  celloidin. 

Some  use  three  strengths  of  celloidin  solution  : — 

(1.)  A thick  syrup  consistence. 

(2.)  One  part  of  (1)  diluted  with  2 vols.  of  ether. 

(3.)  One  part  of  (^2)  diluted  with  2 vols.  of  ether. 

After  thorough  infiltration  the  tissue  has  to  be  embedded.  For 
this  purpose  make  a j)aper  box  (fig.  30)  ; or  use  a pill-box,  or  embed 
it  on  a cork,  thus.  Take  a cork  corresponding 
1 n to  the  size  of  the  object,  roughen  one  end  of 

it,  and  surround  it  with  a piece  of  paper 
fastened  by  a pin  (fig.  31).  Moisten  the 

r roughened  surface  with  absolute  alcohol,  and 
on  it  place  the  tissue  infiltrated  with  celloidin, 
and  surround  the  latter  with  the  thick  solution 
of  celloidin.  Allow  it  to  stand  until  the  cel- 
loidin begins  to  harden  on  the  surface.  This 
takes  place  in  less  than  an  hour.  Place  the 
box  or  cork,  as  the  case  may  be,  in  80  per  cent, 
alcohol  for  24-48  hours,  which  hardens  the 
celloidin  to  such  a consistence  that  it  can  be 
^ ^^Paper  ~ cut  like  a stiff  cheese,  but  the  sections  must  be 

Weight  for  Cel-  ^ut  with  a knife  wetted  with  70  per  cent, 
loidm  with  a lead  t t ^ • i 1 • i • j. 

“ Sinker.”  * alcohol.  it  IS  immaterial  which  microtome  is 

used,  as  long  as  the  knife  is  moistened  with 
not  too  strong  alcohol.  The  sections  may  be  transferred  to  alcohol 
or  water,  and  stained  with  any  suitable  dye.  Some  dyes  stain  the 
celloidin,  especially  the  aniline  dyes,  and  others  do  not. 

A section  after  being  stained  may  be  mounted  in  glycerine  or 
balsam,  but  in  the  latter  case  absolute  alcohol  cannot  be  used  to 
dehydrate  them,  as  celloidin  is  soluble  in  this  fluid.  Alcohol  (95 
per  cent.)  must  therefore  be  used  for  this  purpose.  Moreover,  the 
sections  must  be  clarified  by  origanum  or  bergamot  oil — not  by  oil 
of  cloves,  which  has  a solvent  action  on  the  celloidin — and  mounted 
in  balsam.  (See  also  Clarifying  Eeagents.) 

Objects  embedded  in  celloidin  may  be  kept  ready  for  cutting  foi 
an  indefinite  time  in  75-80  per  cent,  alcohol. 


EMBEDDING. 


4; 


In  cutting  sections  embedded  in  celloidin,  the  knife  must  be  so 
placed  as  to  cut  with  as  much  of  the  blade  as  possible.  The 
hardened  celloidin  is  fixed  to  a piece  of  cork,  which  is  clamped  in 
the  microtome. 

If,  however,  it  be  desired  to  cut  sections  embedded  in  celloidin 
by  the  freezing  method,  the  following  procedure  must  be  adopted. 
After  tlie  tissue  embedded  in  celloidin  has  been  hardened  in  alcohol, 
whereby  it  becomes  not  only  “hardened’^  but  somewhat  milky  in 
appearance,  the  alcohol  must  be  got  rid  of,  which  is  done  by 
keeping  it  in  running  water  for  twenty  four  hours,  when  it  is 
transferred  to  a freezing  mixture  of  gum  and  syrup  (p.  49).  This 
freezing  fluid  gradually  penetrates  the  cheesy-like  mass,  and  dis- 
places the  water.  Such  a preparation  can  be  frozen  in  an  ordinary 
microtome. 

For  embedding  and  cutting  in  celloidin,  the  following  are  the 
stages : — 

(1.)  Organ  hardened,  either  at  first  or  subsequently,  in  absolute 
alcohol. 

(2.)  Then  in  a mixture  of  equal  parts  of  absolute  alcohol  and 
ether  (1-2  days). 

(3.)  In  dilute  celloidin  mixture  (1-5  days). 

(4.)  In  thick  celloidin  mixture  (1-5  days). 

(5.)  Object  placed  on  cork  and  exposed  to  air  to  dry. 

(6.)  Then  in  80  per  cent,  spirit  (i  day). 

(7.)  Cut  sections. 

(8.)  Stain  and  wash  them. 

(9.)  Dehydrate  them  in  96  per  cent,  alcohol  (and  perhaps  1-2 
minutes  in  absolute  alcohol). 

(10.)  Clarify  with  origanum  oil,  xylol,  cedar  or  bergamot  oil. 

(11.)  Mount  in  balsam. 

Suppose  a piece  of  the  human  spinal  cord  is  to  be  embedded  in 
celloidin.  After  being  hardened  properly  it  is  transferred  from 
absolute  alcohol  to  ether,  for  not  more  than  twenty-four  hours.  It 
is  then  placed  for  6-8  days  in  solution  of  celloidin  (3),  in  solution 
(2)  four  or  five  days  and  in  solution  (1)  two  or  three  days.  It  is 
then  embedded  in  celloidin  as  described  above,  and  Avhen  it  begins 
to  harden  by  evaporation  of  the  ether  and  alcohol  it  is  transferred 
to  80  per  cent,  alcohol  for  three  or  four  days,  which  finally  hardens 
it,  but  it  must  be  completely  covered  with  spirit.  If  it  is  to  be 
preserved  for  some  time  before  it'  is  cut,  keep  it  in  70  per  cent, 
alcohol. 

Sections  of  an  organ  embedded  in  celloidin  may  also  be  clarified 
by  means  of  creosote,  or  a mixture  of  i part  of  creosote  and  3 of 
xylol,  which,  however,  must  be  quite  water-free. 


48 


PRACTICAL  HISTOLOGY. 


Scheme  for  Infiltration  and  Embedding  (Bohm  and  Oppel). 
90  per  cent,  alcoliol. 


Staining  in  bulk,  e.g.^  borax  carmine  or  hsematoxylin, 
and  washing  out  the  surplus  stain. 


Absolute  alcohol. 


Toluol  or  turpentine. 
Toluol  or  turpentine-paraffin. 
Paraffin, 
embedding. 


O 

CD 

S 

Ether. 

Celloidin  solution,  No.  3. 
Celloidin  solution,  No.  2. 
Celloidin  solution.  No.  i. 
Embedding. 

80  per  cent  alcohol. 


Object  ready  to  be  cut  into  sections. 


X.— SECTION  CUTTING. 

1.  With  a Razor. — It  is  absolutely  essential  to  have  a sharp 
razor  if  sections  are  to  be  cut  by  hand,  and  it  is  well  that  the 
student  should  practise  this  iTlethod. 

Moisten  the  blade  of  the  razor  with  90  per  cent,  alcohol,  and 
place  some  alcohol  in  an  oblong  glass  vessel.  Dip  the  cutting  blade  or 
razor  into  the  alcohol  after  every  third  or  fourth  section.  Grasp  the 
razor  tightly  with  the  right  hand,  so  that  its  blade  is  horizontal,  its 
edge  directed  to  the  operator,  whose  fingers  are  pressed  against  the 


SECTION  CUTTING. 


49 


back  of  the  blade,  the  back  of  the  hand  being  directed  upwards. 
The  razor  is  made  to  glide  through  the  tissue,  cutting  as  thin 
sections  as  possible,  which  are  placed  in  alcohol.  The  tissue  to  be 
cut  inay  be  embedded  in  any  of  the  ways  already  described,  and  it 
is  held  in  the  left  hand  while  being  cut. 

A razor  ouglit  to  be  sharp  and  free  from  notches  in  its  cutting 
edge.  A razor  with  notches  in  its  edge  causes  striated  bands  in 
the  section.  Notches  can  readily  be  detected  by  drawing  the  razor 
across  one’s  nail  or  over  a piece  of  cardboard.  In  sharpening  the 
razor  use  a soft  hone  moistened  with  water,  then  lay  the  razor  flat 
on  the  hone  and  draw  it  diagonally  from  heel  to  point,  with  the 
edge  forwards. 

The  razor  must  be  frequently  stropped  to  keep  it  sharp,  and  it 
should  not  be  hollow-ground ; it  is  better  to  be  flat  on  one  side. 

2.  Valentin’s  Knife  (fig.  32)  consists  of  two  parallel  blades, 
which  can  be  placed  at  a greater  or  less  distance  from  each  other  by 


Fig.  32.— Valentin’s  Knife. 

means  of  tlie  screw  (a).  The  blades  are  first  set  apart  at  the 
required  distance ; the  thickness  of  the  section  depends  on  the 
distance  between  them.  The  knife  was  formerly  much  used  by 
pathologists  for  making  sections  of  fresh  organs.  It  is  now  rarely 
used. 

3.  Microtomes. — For  many  purposes  some  form  of  freezing 
microtome  will  be  found  most  convenient  for  teaching  purposes ; 
where  a large  number  of  sections  is  required  it  is  indis- 
pensable. 

Preparation  of  Hardened  Tissues  for  Cutting  by  Freezing. — 

In  order  to  secure  the  full  advantages  of  cutting  sections  by  freez- 
ing, the  tissues  must  be  previously  soaked  in  and  saturated  with 
proper  “freezing  fluids.”  If  the  tissues  be  kept  in  alcohol,  first 
remove  all  the  alcohol  by  soaking  them  for  twenty-four  hours  in 
running  water.  After  this  the  tissue  is  soaked  in  gum  mucilage, 
or,  what  is  preferable,  a mixture  of  gum  and  syrup.  The  best 
receipt  is  that  of  Hamilton. 

^lake  a syrup  of  28.5  grams  of  pure  cane-sugar  in  30  cc.  of 
water ; boil  and  saturate  it  with  boracic  acid.  Allow  it  to  cool  and 
filter.  Place  45.6  grams  of  gum  acacia  in  2400  cc.  of  cold  water, 
allow  it  to  dissolve,  saturate  it  with  boracic  acid  by  boiling,  and 
filter. 


6 


D 


PllACTICAL  HISTOLOGY. 


SO 


Freezing  Fluid. 


Take  of  the  syrup  . . . . 4 parts. 

,,  ,,  mucilage  . . . . 5 ,, 

,,  ,,  water  . . . . 9 ,, 

Boil  and  saturate  while  hot  with  boracic  acid.  Filter  through 
muslin. 

The  tissues  are  soaked  for  twenty-four  hours  or  longer  in  this 
fluid,  ^.e.,  after  removal  of  all  alcohol  from  them.  The  longer  they 
are  kept  in  it  the  better ; in  fact,  tissues  may  be  kept  permanently 
ready  for  freezing  in  this  fluid. 

Eutherford’s  Ice-Freezing  Microtome  (fig.  33). — By  means  of  a 
finely-graduated  screw  a brass  plug  can  be  raised  or  lowered  inside 


Tig.  33.— Rutherford’s  Freezing  Microtome  adapted  for  Freezing  with  Ether. 


a brass  cylinder.  At  the  top  of  this  cylinder  is  a stage  or  plate  (B). 
The  plug  has  a small  flattened  brass  knob  screwed  into  it,  so  as  to 
catch  the  frozen  mass,  and  prevent  it  from  being  detached  (K). 
Practically  this  is  the  arrangement  of  the  microtome  devised  by  the 
late  A.  B.  Stirling,  curator  of  the  Anatomical  Museum  of  the 
University  of  Edinburgh.  To  this  arrangement  Professor  Ruther- 
ford added  an  ice-box  (C),  which  surrounds  the  upper  part  of  the 


SECTION  CUTTING. 


SI 


brass  cylinder  or  well.  This  box  is  ])rovided  with  an  exit-tube 
(H),  to  allow  the  water  resulting  from  the  melting  of  the  ice  to 
escape.  The  size  of  the  cylinder  varies  from  1-2  inches  in 
diameter,  but  for  most  purposes  one.  with  a diameter  of  i inch  will 
be  found  sufficient.  The  ice-box  is  covered  on  the  outside  with  a 
thick  layer  of  gutta-percha.  Professor  Hamilton,  of  Aberdeen,  first 
suggested  the  addition  of  a glass  top  to  be  screwed  upon  the  plate 
of  the  instrument. 

By  far  the  most  convenient  cutting  tool  for  use  with  this  micro- 
tome is  an  ordinary  planing-iron  fitted  with  a handle,  as  recommended 
by  Dolepine  (fig.  35). 

Above  all,  the  tissue  must  have  been  properly  hardened,  and 
previously  steeped  in  a freezing  fluid,  either  gum  mucilage  or  gum 
and  syrup,  after  removal  of  all  alcohol  from  it  (p.  50). 

In  using  this  instrument,  screw  the  plug  down  to  the  necessary 
depth,  thus  making  a well  of  the  required  depth — at  least  half  the 
depth  of  the  cylinder — and  into  the  well  drop  a few  drops  of 
glycerine,  or  put  a little  lard  round  the  line  of  contact  of  the  plug 
and  the  cylinder.  This  is  to  prevent  any  of  the  fluid  passing  down 
between  the  plug  and  the  cylinder. 

Fill  the  ice-box  with  a mixture  of  pounded  ice  and  salt,  and 
pack  it  well  around  the  central  brass  cylinder.  Keep  a cork  in 
the  exit  tube  H,  and  only  allow  the  fluid  to  flow  away  when  it 
accumulates  in  large  amount.  In  a short  time  the  temperature  of 
the  plug  is  greatly  reduced.  Pour  into  the  well  a little  mucilage 
(BP),  sufficient  to  form  a layer  al)out  ^ inch  thick,  and  allow  this 
to  freeze.  The  piece  of  tissue  taken  from  the  freezing  mixture  is 
lifted  with  a pair  of  forceps,  and  put  into  the  well,  so  that  it 
touches  and  adheres  to  that  part  of  the  well  farthest  away  from  the 
operator. 

When  the  tissue  is  fixed,  fill  up  the  well  with  mucilage  and 
cover  it  with  a piece  of  sheet  india-rubber,  and  keep  the  latter 
in  position  by  a weight.  This  is  to  prevent  the  entrance  of  the 
freezing  mixture  into  the  well. 

Supposing  the.  tissue  to  be  frozen,  the  operator  seizes  the 
elevating  screw  P with  his  left  hand,  and  in  his  right  holds  the 
planing  iron,  which  is  fixed  in  a wooden  handle.  With  the  left 
hand  the  operator  turns  the  screw,  ^.6^,  elevates  the  tissue,  while  as 
rapidly  as  he  chooses  with  his  right  hand  the  planing  iron,  firmly 
pressed  on  the  glass  plate  at  (about)  an  angle  of  45°,  is  pushed 
rapidly  forwards  and  drawn  backwards,  and  in  a few  seconds 
twenty  or  thirty  sections  accumulate  on  the  upper  surface  of  the 
planing  iron.  By  means  of  a large  camel’s-hair  brush  they  are 
transferred  to  a large  quantity  of  water,  whereby  the  gum  contained 
within  them  is  dissolved  and  the  sections  themselves  uncurl.  One 


52 


PRACTICAL  HISTOLOGY. 


cannot  cut  all  tissues  with  equal  rapidity.  In  the  case  of  many 
tissues  and  organs  (as  elastic  tissue,  lung,  kidney,  &c.),  after  they 
are  completely  frozen,  a quarter  of  an  hour  will  suffice,  if  a leaning 
iron  he  employed,  to  cut  more  than  a thousand  sections.  This, 
however,  cannot  be  done  with  sections  of  the  cerebrum,  cerebellum, 
or  spinal  cord.  With  these  and  with  some  other  organs  it  is 
better  to  cut  each  section  singly. 


1 IG.  34. — Caihcart’s  Freezing  Microtome. 


If  one  has  several  tissues  to  cut,  the  one  tissue  can  be  embedded 
above  the  other  in  the  instrument  at  the  same  time. 

This  instrument,  however,  has  other  advantages,  as  it  can  be 
used  also  as  an  ether-freezing  microtome.  Place  the  tissue, 
previously  saturated  with  the  freezing  fluid,  upon  the  zinc  plate 
Z,  and  cover  it  with  mucilage.  Py  means  of  an  ether-spray 
producer  N,  direct  a spray  of  anhydrous  ether  from  the  bottle  0 
against  the  under  surface  of  the  zinc  plate  Z. 

In  order  to  economise  ether,  any  non-volatilised  is  collected 
and  returned  by  the  tube  T to  the  bottle  0. 


SECTION  CUTTING.  5 3 

In  using  the  instrument  in  this  way,  liowever,  the  piece  of  tissue 
must  be  thin,  not  more  than  5-7  mm.  in  thickness,  or  thereby. 

Cathcart’s  Freezing  Microtome  (fig.  34). — The  instrument  is 
screwed  to  a table  by  means  of  a clamp  (C).  Fix  by  means  of  gum 
the  tissue  to  be  frozen,  not  more  than  i cm.  thick,  upon  the  zinc 
plate.  Fill  the  bottle  with  ether  and  put  the  spray  apparatus 
under  the  zinc  plate.  Work  the  spray  apparatus  until  the  mucilage 
and  tissue  are  frozen. 

Sections  are  cut  by  means  of  a flat  knife  pushed  along  on  the 
glass  supports  (GG), 
the  tissue  being 
raised  by  the  large 
milled  head  placed 
underneath  (^[). 

The  sections  are 
removed  from  the 
knife  by  means  of 
a camers-hair  pen- 
cil, and  placed  in 
water. 

Fig.  35  shows 
Cathcart’s  micro- 
tome clamped  to  a 
table,  and  the 
method  of  cutting 
sections  by  means 
of  a planing  iron. 

The  planing  iron  is 
used  in  exactly  the 
same  way  as  for  Eutherford’s  microtome. 

Roy's  Microtome  (Freezing),  Modified  by  Malassez  (fig.  36). — 
This  instrument  is  extremely  convenient,  the  cutting  instrument 
being  an  ordinary  razor.  Instead  of  ether,  ]\Ialassez  uses  chloride 
of  methyl,  which  is  preserved  in  a stout  metallic  flask.  A stream 
of  the  methyl  chloride  is  directed  against  the  under  surface  of  the  plate 
on  which  the  tissue  to  be  frozen  is  placed.  It  freezes  much  more 
rapidly  than  with  ether. 

Cutting  a Continuous  Series  of  Sections  in  Paraffin. — For  tliis 
purpose  the  “rocking  microtome’’  (fig.  37)  of  the  Cambridge 
Scientific  Instrument  Company,  or  Minot’s  microtome,  is  most 
useful. 

Embed  the  tissue  in  paraffin  in  the  usual  way  ; place  the  razor  in 
position,  and  fix  the  embedded  tissue  to  the  end  of  the  brass  cap  on 
the  horizontal  bar.  Move  the  brass  cap,  with  its  adherent  em- 
bedded tissue,  forwards,  until  it  touches  the  knife-edge.  The 


54  PRACTICAL  HISTOLOGY. 

horizontal  bar  has  an  axle  which  moves  in  a V-shaped  pivot.  At 


Fia.  36.— Malassez’s  Modification  of  Roy’s  Microtome  for  Freezing. 

the  end  of  the  instrument  is  a knobbed  bar,  which  depresses  and 


Fig,  37.  Cambridge  Rocking  Microtome. 

raises  the  horizontal  bar,  and  at  the  same  time  moves  a toothed 


SECTION  CUTTING. 


55 


wheel,  which  is  pushed  round  hy  a small  catch,  wheiehy  the  har 
bearing  the  pivot  is  raised.  At  the  same  time  the  embedded  tissue 
in  paraffin  on  the  horizontal  har  is  brought  down  into  contact  with 
the  edge  of  the  razor. 

The  piece  of  paraffin  should  have  its  sides  squared,  and  the  two 
faces  looking  upwards  and  downwards  should  be  coated  with  soft 
paraffin,  z.e.,  with  a low  melting-point  (48°  C.).  This  is  to  enable 
the  one  section  to  adhere  to  the  other.  On  working  the  instru- 
ment, the  sections  come  off  in  “chains’’  or  ribbons,  and  can  be 
caught  upon  a plate  of  glass.  If  the  sections  tend  to  curl  up,  they 
may  be  “ flattened  ” by  being  placed  in  not  too  warm  water. 

Minot’s  Microtome. — In  this  microtome,  which  is  one  of  the 
best  microtomes  yet  invented  for  embedded  tissues,  as  shown  in  fig. 
38,  the  knife  is  fixed  while  the  embedded  tissue — in  paraffin — fixed 


Eig.  38. — Minot’s  Microtome. 


to  a circular  disc,  is  moved  vertically  upwards  and  downwards  by 
means  of  a wheel.  The  embedded  tissue  is  fixed  to  a disc  wliich 
can  be  moved  around  three  axes,  and  thus  the  tissue  can  be  cut  in 
any  desired  plane.  The  thickness  of  the  sections  is  regulated  by  a 
special  toothed-wheel  mechanism,  which  is  so  arranged  that  sections 


56 


PRACTICAL  HISTOLOGY. 


can  be  cut  varying  from  to  mm.  Instead 

01  the  knife  supplied  with  the  apparatus,  a razor  may  be  used. 

The  seetions  can  be  received  on 
a silk  ribbon,  as  shown  in  fig.  39. 
This  is  clamped  to  the  apparatus. 
The  sections  are  received  on  the 
ribbon,  which  is  rotated  by  means 
of  a milled  head, 

Jung’s  or  Thoma’s  Microtome. 
— In  this  instrument  the  tissue  to 
be  cut  is  fixed  in  a damp,  the 
knife  is  fixed  in  another  heavy 
clamp  which  moves  on  planed  sur- 
faces. After  each  section  the 
tissue  is  pushed  up  an  inclined 
plane  by  means  of  the  milled  head 
on  the  extreme  right  of  the  figure 
(fig.  40). 

Malassez’s  Modification  of 
Roy’s  Microtome  (fig.  41).— Some- 
times it  is  desired  to  cut  sections 
of  a tissue  while  it  is  under  fluid, 
e.p.,  alcohol.  This  can  be  done  as 
shown  in  fig.  41. 

to  move  on  its  base,  and  can  be  placed 


Fia.  39. — Silk  Band  for  Catching  the  Chain 
of  Serial  Sections  made  by  Minot’s 
Microtome. 


The  microtome  is  made 


Fig.  40. — Thoma’s  Sledge  Microtome,  as  made  by  Jung. 


vertically  in  such  a way  that  the  razor  and  the  piece  of  tissue  to  be 
cut  come  to  lie  in  a vessel  filled  with  alcohol. 


SECTION  CUTTING. 


57 


Fig.  41.— Malassez’  Modification  of  Roy’s  Microtome  for  Cutting  Sections  under  a Fluid. 


Fig.  42. --Williams’  Ice-Freeziug  Microtome. 


7 


58 


PRACTICAL  HISTOLOGY. 


Williams’  Freezing  Microtome  (fig.  42). — This  consists  of  a 
wooden,  non-conducting  tub  for  holding  the  freezing  mixture. 
Vertically  in  the  centre  of  this  rises  a brass  cylinder,  into  whose 
upper  end  the  roughened  brass  plate  on  which  the  tissue  is  frozen 
can  be  screwed.  The  lid  of  the  box  is  formed  by  a glass  plate  fitted 


Fig.  43.— Swift’s  Ether-Freezing  Microtome. 


into  a framework  or  kind  of  cap  for  the  tub.  In  the  centre  pf  the 
glass  plate  is  a circular  hole  into  which  the  freezing-plate  projects. 
In  this  instrument  the  tissue  remains  fixed,  while  the  knife  or  blade 
is  depressed  by  the  movement  of  a screw.  The  knife  or  razor  is  fixed 
in  a brass  tripod  frame- work  or  knife-carrier. 


CUTTING  SECTIONS. 


59 


In  using  the  instrument,  fill  the  tubs  with  the  freezing  mixture 
of  ice  and  salt,  and  when  the  central  brass  pillar  becomes  sufficiently 
cooled,  pour  on  it  a little  mucilage,  and  when  this  is  frozen  place  on 
it  the  tissue,  which  must  not  be  more  than  i cm.  in  thickness,  and 
pencil  some  mucilage  on  it.  When  it  is  solid,  the  knife  in  the 
tripod  is  used  to  cut  the  sections ; and  as  the  front  leg  of  the  tripod 
consists  of  a screw,  this  is  turned,  and  thus  the  cutting  edge  is 
brought  to  touch  the  tissue. 

Swift’s  Freezing  Microtome  (fig.  43). — A modification  of  the 
previous  instrument  is  shown  in  fig.  43,  which  is  adapted  for  freezing 
with  ether. 

Hand-Microtome  (fig.  44). — When  onlya  few  sections  are  required, 
this  instrument,  invented  by  Ranvier,  is  extremely  convenient.  The 


Fig.  44,— Ranvier’s  Hand-Microtome. 


tissue  is  embedded  in  the  well  of  the  instrument  in  paraffin  or  elder 
pith,  and  sections  made  by  means  of  a razor,  as  shown  in  the  figure. 
The  tissue  is  gradually  raised  by  means  of  the  milled  head. 

Section-Flatteners. — Sometimes  the  sections  saturated  with 
paraffin  when  cut  exhibit  a great  tendency  to  curl  up.  This  can 
partly  be  avoided  by  pressing  the  section  as  it  is  cut  gently  against 
the  knife,  by  means  of  a camel’s-hair  brush.  Several  section- 
flatteners  attached  to  the  cutting-knife  have  been  used  for  this 
purpose.  Take  a wire  i mm.  in  diameter,  heat  it  in  a flame,  and 
bend  it  twice  at  right  angles,  the  distance  between  the  angles  being 
about  an  inch.  The  free  ends  are  then  bent  round  in  the  form  of 
a hook.  These  hooks  serve  to  fix  the  frame  on  the  back  of  the 
razor,  forming,  as  it  were,  a spring-clip.  The  part  of  the  wire 
between  the  right  angles  is  so  arranged  that  it  lies  parallel  to,  and 
about  one-hundredth  of  an  inch  from,  the  edge  of  the  knife.  In 


6o 


PKACTICAL  HISTOLOGY. 


cutting,  the  section  has  to  pass  between  the  spring  and  the  knife, 
and  is  thus  largely  ])revented  from  curling  up. 

Curled-up  paraffin  sections  may  be  made  to  uncurl  by  being 
placed  in  water  at  about  40°  C.  (Gaskell). 

Cutting  a Continuous  Series  of  Sections  in  Celloidin. — The 
tissue,  embedded  in  celloidin,  is  clamped  in  a microtome,  e,g.^  that 
of  dung,  and  section  after  section  is  made.  The  knife  must  pass  at 
an  acute  angle  through  the  celloidin,  and  must  be  moistened  with 
80  per  cent,  alcohol.  This  is  easily  effected  from  a wash-bottle 
Schanze  of  Leipzig  supplies  such  a bottle  provided  with  a valve, 
which  facilitates  the  outflow  of  a gentle  stream  of  alcohol  upon  the 
cutting  blade.  Weigert’s  method  of  arranging  and  fixing  the 
sections  on  a slide  is  the  best.  Each  section  in  celloidin  as  it  is  made 
is  laid  upon  a narrow  strip  of  curl-paper  by  means  of  a camers-hair 
pencil.  The  curl-paper  is  kept  moist  by  being  placed  on  a plate 
covered  with  blotting-paper  well  moistened  with  80  per  cent,  alcohol. 
The  sections  are  laid  upon  the  curl-paper  in  the  order  desired. 

A slide  is  coated  with  a layer  of  thin  collodion,  and  when  it  is  dry, 
the  celloidin  sections  on  the  curl-paper  are  transferred  to  it.  This 
is  done  by  lifting  up  the  curl-paper,  and  placing  it,  sections  lower- 
most, upon  the  coating  of  collodion  on  the  slide.  Press  on  the 
whole  with  a piece  of  dry  blotting-paper.  The  sections  adhere  to 
the  slide,  and  the  curl-paper  is  removed.  Dry  the  sections  with 
blotting-paper,  and  pour  over  them  a layer,  of  thin  collodion.  They 
are  now  permanently  fixed,  and  can  be  stained  on  the  slide  in  any 
way  that  may  be  desired.  This  is  an  extremely  convenient  method 
for  serial  sections  of  the  central  nervous  system. 


XI —FIXATIVES  AND  SUBSEQUENT  TREATMENT 
OP  SECTIONS. 

Further  Treatment  of  Sections. 

This  depends  on  how  the  sections  have  been  made,  and  whether 
they  have  or  have  not  been  previously  stained.  Paraffin  sections 
must  be  freed  from  paraffin.  If  they  are  unstained,  they  must  be 
stained.  In  most  cases  it  is  found  advantageous  to  fix  paraffin 
sections  to  the  slide  by  means  of  a “fixative.^’  Many  sections  can 
thus  be  fixed  on  one  slide,  and  treated  simultaneously.  The  series 
of  events  will  then  be  for  unstained  paraffin  sections  : — 

(1.)  Fixation  on  a slide. 

(2.)  Removal  of  paraffin. 

(3.)  Staining  the  section. 

(4.)  Mounting  the  specimen. 


FIXATIVES  AND  SUBSEQUENT  TREATMENT  OF  SECTIONS.  6 1 

To  Fix  Paraffin  Sections  on  a Slide.  There  are  several  “fixa- 
tives’’ for  serial  sections,  but  the  following  will  be  found  the 
most  useful. 

(1.)  Collodion  and  Clove-Oil. — Mix  one  part  of  collodion  with 
three  parts  of  clove-oil.  Ey  means  of  a brush  paint  a thin  layer  on 
a slide,  and  on  it  place  the  sections.  Heat  gently  over  die  flame  of 
a lamp,  to  fix  them  firmly  and  drive  off  the  clove-oil. 

(2.)  Albumen  and  Glycerine  [P.  Mayer'). — Mix  filtered  fresh 
white  of  egg  with  an  equal  volume  of  glycerine,  add  a little  car- 
bolic acid  or  morsel  of  thymol  to  prevent  putrefaction.  Mdiite 
of  egg  filters  very  slowly.  A very  thin  layer  is  painted  on  the 
slide,  and  made  smooth  by  means  of  a clean  glass  rod,  which  is 
thus  prepared  to  receive  the  sections.  The  sections  are  flattened 
on  the  albuminised  surface  by  means  of  a fine  brush,  care  being 
taken  that  no  air-bubbles  remain  under  the  sections.  Warm  the 
slide  to  a temperature  just  sufficient  to  coagulate  the  albumen 
(70°  C.).  This  may  also  be  done  by  holding  the  slide  for  a few 
seconds  over  a jet  of  steam. 

Such  substances  as  acids  and  alkalies  which  dissolve  the 
albumen  must  not  be  applied  to  the  sections,  nor  must  the  sections 
be  stained  with  such  substances  as  picrocarmine,  which  also 
dissolve  the  albumen. 

(3.)  Method  of  Gaule. — This  method  depends  on  capillary  attrac- 
tion. The  slide  is  moistened  with  water  or  weak  spirit,  and  on 
this  the  paraffin  sections  are  carefully  spread  out.  The  surplus 
spirit  or  water  is  removed  by  blotting-paper,  and  the  slide  placed 
in  a thermostat  at  50°  C.  for  twenty-four  hours.  Sections  so  dried 
are  heated  for  a moment  above  the  melting-point  of  the  paraffin, 
and  are  then  firmly  fixed  on  the  slide. 

To  Remove  Paraffin  from  the  Sections. — Sections  of  tissues 
soaked  and  embedded  in  paraffin  and  fixed  on  a slide  are  placed  in 
turpentine,  toluol,  or  xylol.  The  extraction  of  the  paraffin  requires 
some  time,  and  takes  place  more  rapidly  when  the  temperature  is 
raised.  The  slides  may  be  fitted  into  a zinc  framework  and 
lowered  into  a bath  of  turpentine  or  toluol.  Clove-oil  must  not  be 
used  if  collodion  and  clove-oil  have  been  used  as  a fixative.  In 
that  case  clarify  with  creosote  and  turpentine.  The  turpentine 
dissolves  out  the  paraffin. 

After  this,  if  the  tissue  has  been  previously  stained  in  bulk, 
before  it  was  embedded,  drive  away  the  turpentine  with  xylol  or 
clove-oil,  and  mount  the  section  in  balsam. 

If,  however,  the  sections  are  from  an  unstained  tissue,  after 
dissolving  out  the  paraffin  Avith  turpentine,  the  latter  must  be 
displaced  by  absolute  alcohol,  and  the  slides  are  passed  through 
alcohols  of  various  strengths  and  then  into  water,  f.c.,  provided  the 


62 


PRACTICAL  HISTOLOGY. 


sections  are  to  be  stained  in  a watery  solution  of  a dye.  The 
sections  are  then  coloured  in  situ  on  the  slide.  If  the  sections  are 
to  be  mounted  in  balsam^  they  must  go  through  the  same  process  in 
the  reverse  order,  viz.,  increasing  strengths  of  alcohol — a clarifying 
agent,  clove-oil,  or  xylol — and  finally  they  are  mounted  in  balsam. 


Scheme  for  the  Further  Treatment  of  Paraffin  Sections. 
Paraffin  section. 


Fixation  on  slide. 


Toluol  or  turpentine. 


STAINING  REAGENTS. 


63 


XIL— STAINING  REAGENTS. 

Staining. — This  process  depends  on  the  fact  that  different  tissues, 
or  different  parts  of  the  same  tissue,  have  an  affinity  for  certain  dyes, 
and  not  for  others.  Thus  some  dyes  stain  only  the  nuclei,  others 
however  may  cause  a uniform  stain,  ail  the  tissues  being  of  the  same 
colour.  By  using  some  decolorising  reagent,  it  is  possible  to  remove 
the  stain  from  certain  parts  of  the  preparation,  leaving  other  parts 
stained. 

A thin  section  of  a tissue  or  an  organ,  as  a rule,  when  examined 
shows  but  little  differentiation  of  its  several  parts.  Only  in  cases 
where  pigment  is  naturally  present  is  this  difference  very  marked. 
Some  substances  when  applied  to  the  section  stain  one  part  and 
leave  other  parts  unaffected,  thus  enabling  one  to  differentiate  more 
easily  the  several  parts  of  a section. 

Those  substances  which  stain  the  nuclei  chiefly  have  been  called 
nuclear  stains.  The  section  is  placed  in  a weak  solution  of  the  dye, 
e.g.,  hsematoxylin ; and  after  it  seems  to  be  sufficiently  stained,  the 
surplus  dye  is  removed  by  thoroughly  washing  the  section  in  water 
cr  alcohol,  a part  of  the  dye  remaining  united  with  the  chromatin  of 
the  nucleus  and  colouring  the  latter.  Such  stains  may  also  colour 
to  a less  degree  some  other  parts  of  the  section.  Amongst  nuclear 
stains  are  carmine,  hsematoxylin,  and  some  of  the  aniline  colours. 

When  a section  is  stained,  it  is  called  Section  Staining,  but  the 
tissue  may  be  stained  in  bulk  before  the  sections  arc  made  (p.  44) 
— staining  in  bulk. 

A.  Carmine  and  its  Compounds. 

Carmine. — In  order  to  obtain  a strong  solution  of  this  dye, 
certain  solvents  require  to  be  employed.  It  is  readily  soluble  in 
ammonia,  yielding  an  ammoniacal  solution,  which  may  be  made 
strong  or  weak.  The  ammoniacal  solution  may  be  diluted  to  any 
extent  required  with  water,  and  practically  the  best  results  are 
obtained  by  allowing  sections  to  remain  for  a long  time  (24-48 
hours)  in  a weak  solution. 

1.  Strong  Ammoniacal  Carmine  Solution. — Rub  up  in  a mortar 
2 grams  of  pure  carmine  with  a few  drops  of  water,  add  5 cc.  of 
strong  liquor  ammoniae,  mix  thoroughly,  and  add  100  cc.  of  water. 
Place  the  whole  in  a bottle,  and  after  a day  or  so  any  undissolved 
carmine  is  filtered  off  and  the  clear  fluid  kept  as  a stock  solution. 
This  solution  may  be  diluted  to  any  required  extent.  If  it  smell 
strongly  of  ammonia,  the  excess  of  ammonia  must  be  allowed  to 
evaporate.  When  the  solution  becomes  neutral  it  is  very  liable 
to  undergo  putrefaction,  but  this  may  be  avoided  by  placing  a 
small  piece  of  thymol  in  it  to  preserve  it. 


64 


PKACTICAL  HISTOLOGY. 


2.  Frey’s  Carmine. — ^Ordiiiary  carmine  has  two  drawbacks  : it 
is  apt  to  undergo  putrefaction,  and  as  tlic  ammonia  escapes  the 
carmine  is  precipitated. 

Carmine  . . . . .0.3  gram. 

Distilled  water  . . . . . 30  cc. 

Dissolve  the  carmine  in  the  water,  adding  ammonia  drop  by  drop 
until  solution  is  complete.  Then  add — 

Glycerine  . . . . . 30  cc. 

Alcohol  . . . . . 4 ,, 

and  shake  the  mixture.  Keep  it  in  a stoppered  bottle.  It  has 
no  advantage,  as  far  as  coloration  is  concerned,  over  ordinary 
carmine,  but  it  can  be  kept  for  a long  time  unchanged. 

3.  Alcoholic  Borax  Carmine  {Grenadier). 

Carmine  ......  3 grams. 

Borax  . . . . . . 4 ,, 

Water  ......  100  cc. 

Dissolve  the  borax  in  the  water  and  add  the  carmine,  which  is 
quickly  dissolved,  especially  with  the  aid  of  gentle  heat.  Add  100 
cc.  of  75  per  cent,  alcohol,  and  filter. 

4.  Watery  Borax-Carmine. — Kub  up  in  a mortar  8 grams  borax 
with  2 grams  carmine,  and  add  150  cc.  water.  After  twenty-four 
hours  decant  and  filter.  Tissues  to  be  stained  in  bulk — e.g,^  after 
hardening  with  corrosive  sublimate — are  placed  for  twenty-four 
hours  or  longer  in  this  fluid.  They  are  then  transferred  to  acid 
alcohol  (i  per  cent.  HCl  in  70  per  cent,  spirit  for  twenty-four 
hours),  and  then  into  alcohol. 

If  it  be  desired  to  stain  in  bulk  without  bringing  the  tissue  into 
contact  with  water,  then  use  : — • 

5.  Carmine-Solution  (P.  Mayer). — For  staining  in  bulk,  and  also 
for  sections,  Mayer  recommends  the  following  : — Suspend  4 grams 
carmine  in  15  cc.  water,  and  then  add  30  drops  hydrochloric  acid, 
gently  heating  the  mixture.  Add  95  cc.  alcohol  (85  per  cent.)  and 
boil.  Neutralise  with  ammonia  and  on  cooling  filter. 

6.  Borax  Carmine  {Grenadier). 

Carmine  .....  i gram. 

Borax  ......  2 grams. 

Distilled  water  .....  200  cc. 

The  borax  dissolves  the  carmine.  The  whole  is  placed  in  a 
porcelain  capsule  and  heated  to  boiling,  when  the  fluid  becomes  of 
a dark-purplish  or  bluish-red.  Add  a few  drops  of  5 per  cent, 
acetic  acid,  until  the  colour  becomes  more  like  that  of  carmine 
dissolved  in  ammonia.  Let  it  stand  for  twenty-four  hours,  and 


STAINING  REAGENTS. 


65 


filter.  Add  a drop  or  two  of  carbolic  acid  to  preserve  it.  This 
gives  a diffuse  stain,  so  that  the  sections  have  to  be  treated  with 
acid  alcohol  (p.  65).^ 

The  original  receipt  is 


Carmine 
Borax 
Water  . 

Alcohol  (70  per  cent.) 


.5  to  .75  gram. 

2 „ 

100  cc. 
100  ,, 


Borax-carmine  is  chiefly  used  for  staining  tissues  “in  bulk.” 
Small  pieces  of  tissue,  to  ^-inch  cubes  or  larger,  may  be  left  in  it 
for  days,  and  they  do  not  become  over-stained.  It  gives  by  itself 
a diffuse  stain ; hence  to  get  its  effect  concentrated  upon  the  nuclei, 
for  which  it  has  a special  affinity,  the  pieces  of  tissue  must  be  placed 
for  twenty-four  hours  or  thereby  in  70  per  cent,  alcohol  containing 
I per  cent,  of  hydrochloric  acid. 

Acid  Alcohol. — This  is  called  acid  alcohol,  and  is  prepared  thus — 


Hydrochloric  acid  . . , . i cc. 

Alcohol  . . . . . • 70  j) 

Water  . . . . . • 30 

When  tissues  are  placed  in  the  acid  alcohol,  they  change  in 
colour  to  a bright  scarlet.  A certain  amount  of  the  surplus 
carmine  is  extracted,  but  the  nuclei  become  intensely  stained. 

This  method  is  particularly  valuable  for  a large  number  of  organs, 
and  especially  where  nuclear  staining  is  desired. 

7.  Alum  Carmine. — Dissolve  5 grams  of  p>otash-alum  in  100  cc. 
water.  Add  i gram  carmine,  and  boil  for  a quarter  of  an  hour. 
Make  up  the  bulk  with  water  and  filter.  Add  a drop  of  carbolic 
acid  to  preserve  it,  as  fungi  rapidly  form  in  it.  It  has  the  advan 
tage  of  not  over-staining  tissues  left  in  it  for  a long  time. 

8.  Lithium  Carmine  {Orth). 

Carmine  . . . . *2.5  grams. 

Saturated  solution  of  lithium  carbonate  . 100  cc. 


Dissolve  the  carmine  in  the  cold  saturated  solution  of  lithium 
carbonate ; solution  occurs  very  quickly.  It  gives  a diffuse  stain, 
to  nearly  all  tissues  very  rapidly,  and  the  sections  must,  therefore, 
be  transferred,  without  previous  washing  in  water,  to  acid  alcohol 
(p.  65).  They  can  then  be  mounted  in  glycerine  or  balsam  as 
desired.  The  nuclei  are  stained  a brilliant  red.  It  cannot  be  used 
for  sections  fixed  on  a slide  by  means  of  white  of.  egg. 

Application. 

(1.)  Stain  (2-3  minutes). 

(2.)  Wash  out  surplus  dye  in  acid  alcohol  (^-i  minute),  ^.e.,  in  100 
cc.  of  70  per  cent,  spirit  + i cc.  HCl. 

(3.)  Remove  all  acid  by  prolonged  washing  in  water. 

(4.)  Alcohol,  oil,  balsam. 

^ Archiv  f.  Mik.  Anat.,  vol.  xvi.  p.  363. 


E 


66 


PRACTICAL  HISTOLOGY. 


9.  Picro-Lithium  Carmine. — This  is  even  preferable  to  the  fore- 
going, because,  in  addition  to  staining  nuclei  red,  it  stains  certain 
other  parts  yellow. 

Lithium  carmine  . . . . . 50  cc. 

Saturated  solution  of  picric  acid  . . . 100  ,, 

Mix  the  two  slowly.  If,  after  trying  it,  one  or  other  colour  is 
too  pronounced,  add  a little  more  of  the  other. 

The  sections  are  to  be  treated  with  acid  alcohol  like  the  fore- 
going. The  acid  alcohol,  however,  ultimately  extracts  the  picric 
acid.  This  is  avoided  by  not  leaving  them  too  long  in  acid 
alcohol.  If  the  picric  stain  be  removed,  it  may  be  restored  at  once 
by  dipping  the  section  in  absolute  alcohol  to  which  a little  picric 
acid  has  been  added. 

10.  Picro-Carmine. — This  most  valuable  reagent  was  introduced 
by  Ranvier,  and  has  the  great  advantage  of  giving  a double  stain 
without  the  use  of  acid  or  alkali. 

(a.)  Banvier^s  method  of  preparing  it  is  as  follows : — To  a 
saturated  watery  solution  of  picric  acid  add  a saturated  ammoniacal 
solution  of  carmine  until  precipitation  just  appears,  i.e.^  until  satura- 
tion. The  fluids  must  be  well  mixed.  Leave  it  exposed  in  shallow 
vessels  to  crystallise,  but  protect  it  from  the  dust.  Crystals  are 
deposited,  and  also  some  amorphous  carmine.  After  several  weeks, 
when  its  bulk  is  reduced  to  one-third,  decant  the  liquid,  filter,  and 
evaporate  it  to  dryness  on  a water-bath.  Redissolve  it  and  the 
crystalline  deposit  in  water,  filter,  and  evaporate  to  dryness.  The 
brown  powder  so  obtained  is  dissolved  in  the  proportion  of  i per 
cent,  in  water.  This  fluid,  prepared  in  this  way,  gives  very  satis- 
factory results. 

(h.)  Stohr^s  Method. — A very  good  solution  is  obtained  by  this 
method.  To  50  cc.  water  add  5 cc.  liquor  ammonise  and  i gram 
carmine,  which  is  rapidly  dissolved.  After  complete  solution,  add 
50  cc.  of  a saturated  solution  of  picric  acid.  Set  the  mixture  aside 
for  two  or  three  days  in  a large  open  flat  evaporating  dish,  and  after 
this  time  filter.  To  the  fljtrate  add  a drop  of  chloroform  to  prevent 
the  formation  of  fungi. 

Precautions. — Preparations  stained  with  picro-carmine  are  not 
liable  to  be  over-stained,  and  may  be  mounted  in  Farrant’s  solution 
or  glycerine  acidulated  with  formic  acid  (i  per  cent.)  [formic  acid 
sp.  gr.  1. 16]. 

In  staining  sections  with  picro-carmine,  cover  the  section  with 
picro-carmine,  and  after  a few  minutes  remove  the  surplus  pigment. 
On  no  account  should  the  section  he  jdaced  in  water.  Water  rapidly 
extracts  the  picric  acid  and  leaves  the  preparation  stained  with  the 
carmine  only.  Add  a drop  of  Farrant’s  solution  or  formic  glycerine, 


STAINING  REAGENTS. 


67 


and  cover.  Such  sections  improve  with  keeping,  and  the  surplus 
picro-carmine  is  really  an  advantage,  for,  after  a time,  the  nuclei 
become  more  differentiated — red,  and  other  parts  yellow. 

If  it  be  desired  to  mount  sections  stained  wdth  picro-carmine  in 
balsam,  the  alcohol  through  w^hich  they  are  passed,  or  the  clove-oil, 
must  contain  some  picric  acid  to  restore  the  yellow  colour. 

11.  Carmine  and  Dahlia  Fluid  ( Wedphal). — Dissolve  i gram  of 
carmine  in  2.5  per  cent,  of  alum.  Take  of  this — 

Alum  carmine  . . . . ^ . 100  cc. 

Glycerine  .....  100  ,, 

Sat  sol.  of  dahlia  in  absolute  alcohol  . . 100  ,, 

Acetic  acid  . . . . . 20  ,, 

This  fluid  is  specially  useful  for  staining  the  granular  cells 
(‘‘Mastzellen  ”)  of  the  liver. 

12.  Cochineal  (Csokor). 

Powdered  cochineal  . . . • 5^  grams. 

Alum  . . . . . • 5 

Water  ......  500  cc. 

Dissolve  the  alum  in  the  water,  add  the  cochineal  (Coccus  cacti), 
and  boil.  Evaporate  down  to  two-thirds  of  its  original  volume. 
Filter.  Add  a few  drops  of  carbolic  acid  to  prevent  the  formation 
of  fungi.  This  is  an  excellent  nuclear  stain,  especially  for  the 
central  nervous  system. 

It  stains  nuclei  of  a violet-red  and  does  not  overstain,  so  that 
sections  may  be  left  in  it  for  many  hours.  It,  however,  does  not 
stain  well  objects  that  naturally  stain  with  difficulty. 

Application. 

(1.)  Stain  for  an  hour  or  more. 

(2.)  Wash  in  water. 

(3.)  Alcohol,  oil,  balsam. 

13.  Indigo  Carmine  (Merkel), 

Solution  A. — Carmine  . 

Borax 
Water 

Solution  B. — Indigo  carmine 
Borax 
Water 

Keep  the  solutions  separate  (B  is  apt  to  develop  a precipitate). 
When  required,  mix  equal  volumes  of  A and  B.  The  mixture 
undergoes  a change  within  a week,  and  hence  it  is  better  to  make 
it  fresh  Avhen  required.  Sections  must  remain  in  it  at  least  twenty- 
four  hours — with  advantage  longer — but  the  results  are  certainly 
satisfactory.  The  stained  sections  are  placed  for  half-an-hour  or 


2 grams. 
8 „ 

1 30  cc. 

8 grams. 
8 „ 
130  cc. 


68 


PRACTICAL  HISTOLOGY. 


thereby  in  a saturated  solution  of  oxalic  acid,  which  extracts  the 
superfluous  pigment.  Mount  sections  in  Tarrant’s  solution  or 
balsam.  (See  Stomach.) 

B.  Hsematoxylin  and  Logwood. 

Hsematoxylin. — This  substance  was  introduced  to  the  notice  of 
histologists  by  Bohmer.  It  is  one  of  the  most  valuable  nuclear 
staining  reagents  we  possess,  and  this  is  specially  the  case  when  its 
violet-blue  stain  is  set  ofi*  by  contrast  with  a ground  stain  of  eosin, 
picric  acid,  or  other  appropriate  dye. 

1.  Hsematoxylin  [Rohmer). — Make  a solution  containing — 

Hfematoxyliii  .....  i gram. 

Absolute  alcohol  . . . , . loo  cc. 

Make  a second  solution  of — 

Alum  . . . . . - 5 grams. 

Distilled  water  . . . . . loo  cc. 

Add  drop  by  drop  the  first  solution  to  a little  of  the  second  until 
a deep-violet  colour  is  obtained.  The  fluid  is  placed  for  fourteen 
days  in  an  open  vessel,  protected  from  dust,  and  exposed  to  the 
light  and  air,  when  it  becomes  of  a bluish  tint.  This  fluid  is  said 
to  “ ripen.”  Oxidation  processes  take  place  whereby  the  hsemato- 
xylin is  converted  into  hamatein.  Filter.  Add  a fragment  of 
thymol  to  preserve  it.  It  stains  tissues  in  5-15  minutes. 

It  is  sufficient  to  make  a saturated  solution  of  the  crystals  of 
hsematoxylin  in  a small  quantity  of  absolute  alcohol.  Add  a few 
drops  to  a I per  cent,  solution  of  alum,  which  yields  a light  violet- 
coloured  fluid.  Expose  the  fluid  to  light  and  air,  when  it  becomes 
blue.  It  is  well  to  prepare  this  fluid  several  weeks  beforehand  to 
enable  it  to  ‘‘  ripen.” 

The  ordinary  manipulative  procedure  for  hsematoxylin  staining  is 
as  follows : — 

(1.)  Stain  the  section  (2-10  minutes). 

(2.)  Wash  in  distilled  water. 

(3.)  Allow  section  to  remain  12-24  kours  in  distilled  water. 

(4.)  Eemove  water  with  alcohol, — add  ethereal  oil,  balsam. 

Bdhmer’s  hsematoxylin  is  well  adapted  for  staining  sections.  It 
is  best  adapted  for  tissues  hardened  in  sublimate,  alcohol,  picric  or 
nitric  acid,  and  not  so  good  for  those  from  chromium  salts  or  osmic 
acid.  Sections  to  be  stained  with  it  before  being  placed  in  the  dye 
are  better  to  be  placed  first  in  water  or  in  i per  cent,  alum  solution. 
If  taken  direct  from  alcohol  they  may  contain  a deposit  after  being 
stained.  After  staining,  the  sections  are  thoroughly  washed  in 
ordinary  water.  Hsematoxylin  stains  the  chromatin  of  the  nuclei 


STAINING  REAGENTS. 


69 


a deep  blue,  and  other  parts  of  some  tissues  light  blue,  e.^.,  the 
matrix  of  hyaline  cartilage. 

2.  Strong  Nucleus-Staining  Hsematoxylin  {Hamilton), 


Ha^matoxylin  . 
Alum 
Glycerine 
Distilled  water  . 


12  grams. 

50  n 
65  cc. 

130 


Boil,  and  while  hot  add  5 cc.  liquid  carbolic  acid.  Allow  the 
mixture  to  stand  in  the  sunlight  for  at  least  a month. 

3.  Delafield’s  Haematoxylin. — To  100  cc.  of  a saturated  solution 
of  ammonia  alum  add  drop  by  drop  a solution  of  i gram  haematoxylin 
dissolved  in  6 cc.  absolute  alcohol.  Expose  to  the  air  and  light 
for  a week.  Filter.  Add  25  cc.  glycerine  and  25  cc.  of  methylic 
alcohol.  Allow  it  to  stand  exposed  to  the  light  for  a long  time. 
Filter. 

This  solution  stains  extremely  rapidly,  and  may  be  greatly  diluted 
when  it  is  used.  It  keeps  for  a very  long  time,  and  stains  well 
even  tissues  which  have  been  hardened  in  chromic  or  osmic  acid. 

4.  Kleinenberg’s  Haematoxylin. — (i.)  Make  a saturated  solution 
of  calcium  chloride  in  70  per  cent,  alcohol.  Shake  it  well,  and 
allow  it  to  stand.  Decant  the  saturated  solution,  add  alum  to 
excess,  shake  it  well,  allow  it  to  stand  for  a day  or  so,  and  filter. 

(2.)  Make  a saturated  solution  of  alum  in  70  per  cent,  alcohol. 
Filter. 

(3.)  To  one  volume  of  the  filtrate  from  (i)  add  eight  volumes 
of  (2). 

(4.)  To  (3)  add  drop  by  drop  a saturated  solution  of  haematoxylin 
in  absolute  alcohol,  until  it  becomes  of  a decided  purple  colour, 
but  not  too  dark,  as  the  solution  becomes  darker  by  keeping  and 
exposure  to  light.  It  should  be  prepared  at  least  a few  months 
before  it  is  wanted. 

It  may  be  diluted  to  any  extent  by  adding  the  mixture  (i)  or 
(2).  As  it  contains  much  spirit,  sections  placed  in  it  must  be 
covered,  protected  from  evaporation,  else  the  spirit  will  rapidly 
evaporate. 

This  logwood  stain  is  particularly  valuable  when  it  is  required 
to  stain  a tissue  or  an  organ  ‘‘  in  bulk.^’  This  a diluted  solution 
will  do  in  24-48  hours,  provided  the  pieces  be  not  too  large. 

5.  Acid  Haematoxylin  {Ehrlich). 

Haematoxylin  .....  i gram. 

Absolute  alcohol  . , , . 30  cc. 

To  the  solution  add — 


Glycerine 
Distilled  water  . 
Glacial  acetic  acid 


60  cc.  \ Saturated 
60  ,,  J with  alum. 
3 „ 


70 


PRACTICAL  HISTOLOGY. 


Dissolve  the  haematoxylin  in  the  alcohol,  add  the  glycerine  and 
water,  and  then  the  acid.  At  first  the  solution  is  light-red,  hut 
wlien  it  has  been  exposed  to  the  air  for  2-3  weeks  it  gets  bluish. 
It  does  not  over-stain,  and  the  tissues  stained  with  it,  Avhen  exposed 
to  the  light,  become  violet  or  bluish  tinted.  It  may  be  used  either 
for  sections  or  for  staining  in  hulk,  and  in  the  latter  case  it  does 
not  tend  to  over-stain. 

Application. — Sections  from  alcohol — 

(I.)  Stain  (3-5  minutes). 

(2.)  Wash  in  alcohol  (90  per  cent.). 

(3.)  Alcohol,  oil,  balsam. 

Garhini  finds  it  better  to  place  the  sections  after  staining  in — 

(2.)  Distilled  water. 

(3.)  Solution  of  carbonate  of  lithia  (.2K  per  cent.). 

(4.)  Alcohol. 

6.  Glycerine  Haematoxylin  {Renaut), — Saturate  perfectly  neutral 

glycerine  with  potash-alum,  and  to  it  add  drop  by  drop  a saturated 
solution  of  haematoxylin  in  90  per  cent,  alcohol,  until  a deep-violet 
tint  is  obtained.  About  one-fourth  part  of  the  haematoxylin  solution 

has  to  be  added.  Let  it  stand  exposed  to  the  light  for  weeks,  and 

filter. 

7.  Eosin  Haematoxylin  {Renaut). — Add  drop  by  drop  a con- 
centrated watery  solution  of  eosin  to  200  cc.  of  glycerine  saturated 
with  potash-alum.  Filter.  Add  drop  by  drop  an  alcoholic  solution 
of  haematoxylin.  Expose  to  light  for  weeks,  and  filter. 

8.  Heidenhain’s  Haematoxylin. 

(1.)  This  is  used  for  staining  in  hulk.  Prepare  J per  cent, 
watery  solution  of  haematoxylin,  which  must  not  be 
kept  too  long.  Boil  the  haematoxylin  in  water  and 

allow  it  to  cool.  Place  the  hardened  tissue  in  this  dye 

for  24-48  hours. 

(2.)  Transfer  it  for  24  hours  to  .5  per  cent,  watery  solution 
of  yellow  chromate  of  potash  (24-48  hours).  This 
causes  dark  clouds  in  the  fluid,  so  that  the  chromate 
must  he  frequently  changed. 

(3.)  Wash  carefully  in  water.  The  tissues  are  then  hardened 
in  alcohol,  and  may  be  embedded  in  paraffin.  It  is  best 
adapted  for  objects  hardened  in  absolute  alcohol,  e.g., 
salivary  glands,  pancreas  (Lesson  XXIII.),  or  in  picric 
acid.  Besides  tinting  the  nuclei  a greyish-blue,  the 
protoplasm  of  the  cells  has  a fine  steel-gray  tint,  but 
the  sections  must  not  be  too  thick. 


STAINING  REAGENTS. 


71 


9.  Hamatein.  It  is  well  known  that  a solution  of  hseinatoxylin 
(CjgHj^Og)  after  being  prepared  must  stand  some  time  to  ‘‘  ripen  ’’ 
before  it  is  ready  for  use.  The  substance  ultimately  formed  chiefly 
by  the  action  of  the  air  is  hamatein  (CjgHj2^6)>  which  occurs  in 
commerce  in  the  form  of  a brown  powder  which  is  soluble  in  alcohol 
or  water.  P.  Mayer  recommends  the  following  solution  of  this 
body  in  alum  or  Ham-alum  : ^ — 


(«.)  Hamatein  .... 

. I gram. 

Alcohol  (90  per  cent.) 

50  cc. 

Dissolve  by  heating. 

(&.)  Alum  .... 

50  grams. 

Distilled  water 

. 1000  cc. 

Mix  the  fluids  (a)  and  (h).  Allow  the  mixture  to  settle,  and  use 
the  clear  supernatant  fluid  as  a stain.  If  it  be  too  strong,  dilute 
with  distilled  water,  or,  better  still,  with  alum  solution. 

I have  used  this  extensively  during  the  last  year  for  staining  in 
bulk,  and  find  it  to  be  an  excellent  dye. 

Logwood. — Staining  solutions  were  formerly,  and  sometimes 
are,  made  from  logwood  chips. 

10.  Logwood  [MitchelVs). — The  tannic  acid  is  removed. 

Place  finely-ground  logwood  (2  oz.)  in  a funnel ; pack  it  well, 
and  allow  water  to  percolate  through  it  until  it  flows  away  with 
but  little  colour.  Allow  the  water  to  drain  away ; spread  the 
logwood  on  a board  to  dry. 

Dissolve  alum  (9  oz.)  in  8 oz.  of  water.  Moisten  the  dry  logwood, 
pack  it  again  tightly  into  a funnel,  and  pour  on  the  alum  solution. 
Close  the  lower  end  of  the  funnel,  and  allow  the  alum  solution  to 
extract  the  dye  from  the  logwood  for  forty-eight  hours. 

Allow  the  coloured  fluid  to  flow  off,  and  percolate  4 oz.  of  water 
through  the  logwood  in  the  funnel.  Add  a few  drachms  of  glycerine 
and  rectified  spirit.  Dilute  largely  when  using,  so  that  -staining 
will  take  place  slowly. 

11.  Other  solutions  of  hsematoxylin,  including  Weigert's^  are 
referred  to  in  the  text.  (Lessons  on  Central  Nervous  System  and 
Salivary  Glands.) 

General  Statement  regarding  Haematoxylin. — Hsematoxylin  is 
specially  useful  for  staining  tissues  hardened  in  chromic  acid. 
Ehrlich’s  hsematoxylin  is  much  to  be  commended.  In  all  cases  it 
is  better  to  clarify  sections  stained  with  hsematoxylin  by  means  of 
xylol — not  clove-oil — before  mounting  in  balsam. 

Haematoxylin  and  logwood  are  amongst  the  best  nuclear  stains 
we  possess,  and  the  tissues  which  stain  best  are  those  hardened  in 
alcohol ; but  those  also  from  Muller’s  fluid  stain  well.  With  those 

^ Zeitsch,/.  wiss,  Mik.^  viii.  p.  341. 


72 


PRACTICAL  HISTOLOGY. 


hardened  in  watery  solutions  of  chromic  acid  it  is  otherwise.  It  is 
sometimes  very  difficult  to  get  them  to  stain.  This  may  sometimes 
be  effected  by  soaking  the  sections  previously  in  a dilute  solution  of 
sodic  carbonate. 

They  are  also  very  valuable  in  double  and  treble  staining. 

In  the  case  of  preparations  stained  by  hsematoxylin  or  logwood, 
over-staining  may  be  got  rid  of  by  placing  the  sections  in  dilute 
acetic  acid.  This  will  rapidly  extract  the  surplus  stain,  but  at  the 
same  time,  if  allowed  to  act  too  long,  it  will  make  the  remaining 
part  red.  Great  care  should  bo  taken  afterwards  by  thorough 
washing  of  the  sections  in  water  to  remove  every  trace  of  the  acid. 

C.  Eosin. 

1.  Eosin. — This  substance  is  readily  soluble  in  alcohol  and  water. 
Make  a 5 per  cent,  watery  solution.  It  gives  a beautiful  diffuse 
rosy  hue,  and  stains  very  quickly,  in  a minute  or  two.  The 
stronger  solution  can  be  diluted  as  required.  It  forms  one  of  the 
best  ground-stains  in  contrast  to  logwood  or  one  of  the  numerous 
aniline  dyes.  When  using  it  as  a double  stain,  logwood  and 
eosin,  stain  the  section  first  of  all  in  logwood,  and  if  it  is  to  be 
mounted  in  balsam,  clarify  with  clove-oil  in  which  a little  eosin  has 
been  dissolved.  Sections  stained  with  it  can  be  mounted  in  balsam, 
Farrant’s  solution,  or  glycerine.  It  is  a specific  stain  for  the 
haemoglobin  of  red  blood-corpuscles,  as  it  stains  it,  even  after 
hardening  in  chromic  salts,  a copper-red  colour,  while  it  also  stains 
the  granules  of  certain  leucocytes  of  the  blood  of  a reddish  tint.  It 
is  used  very  extensively,  and  in  a very  dilute  solution  is  a good 
stain  for  cartilage  and  striped  muscle. 

D.  The  Aniline  Dyes. 

Watery  or  alcoholic  solutions  of  the  aniline  dyes  stain  sections 
with  great  rapidity.  The  word  ‘‘stain”  is  perhaps  not  quite  the  right 
word  to  use.  It  is  rather  a process  of  imbibition  than  staining 
proper.  One  of  the  difficulties  in  using  aniline  dyes  is  the  rapidity 
with  which  sections  have  to  be  transferred  from  one  liquid  to  another. 
They  are  not  used  for  staining  in  bulk.  After  staining  and  dehydrat- 
ing, it  is  best  to  clarify  the  sections  (except  in  special  cases)  with 
cedar  or  bergamot  oil  or  xylol.  The  sections  are  mounted  in  balsam, 
not  in  glycerine,  as  the  latter  dissolves  the  dyes. 

They  are  amongst  the  most  valuable  so-called  staining  reagents 
we  possess,  and  although  many  of  them  do  not  yield  permanent 
preparations — the  colour  fading  after  a time — still  the  results 
obtained  by  their  use  are  so  important  that  it  behoves  the  student 
to  use  them  frequently.  It  is  most  important  that  they  should  be 
obtained  from  reliable  sources. 


STAINING  REAGENTS. 


73 


The  aniline  colours  are  divided  by  Ehrlich  into  acid,  basic,  and 
neutral  compounds.  Of  the  three,  the  basic  colours  are  most  used, 
as  they  are  excellent  nuclear  stains.  Some  of  them  have  special 
affinities  for  certain  tissues,  and,  as  is  well  known,  they  are  of  the 
utmost  value  in  bacteriological  investigations.  They  may  be  kept 
in  drop  bottles.  All  of  them  are  soluble  in  alcohol,  and  most  of 
them  in  \Vater ; and  so  powerful  are  they,  that  usually  a i per  cent., 
or  even  a much  weaker  solution,  suffices  to  stain  tissues  in  a few 
minutes.  Some  of  them,  according  to  Ehrlich’s  researches,  stain 
better  when  they  are  mixed  with  a mordant. 

Aniline-Oil  and  Aniline-Water  {EkrllclCs  Method). — Shake  up 
excess  of  pure  aniline-oil  with  excess  of  water,  and  allow  it  to  stand. 
The  most  of  the  oil  sinks  to  the  bottom.  This  solution  sliould  not 
be  kept  too  long;  in  fact,  it  is  better  to  make  it  fresh.  Filter  a 
little  of  the  aniline-water  into  a watch-glass.  To  the  fluid  in  the 
watch-glass  add  ten  or  twelve  drops  of  a concentrated  alcoholic 
solution  of  any  of  the  aniline  dyes  it  is  desired  to  use  in  this  way. 

Very  few  of  the  preparations  stained  by  aniline  dyes  can  be  pre- 
served in  glycerine  or  Earrant’s  solution.  A 50  per  cent,  solution 
of  acetate  of  potash  keeps  their  colours  well,  but  it  is  a medium 
which  it  is  difficult  to  keep  tightly  under  the  cover-glass.  Many  of 
them  can  be  preserved  in  balsam,  but  most  of  them  must  be  kept 
away  from  the  action  of  acids.  In  a few  cases  the  colour  is  partly 
extracted  and  fixed  with  dilute  acids,  but  in  such  cases  the  free  acid 
must  be  removed  from  the  section  before  it  is  finally  mounted. 

For  convenience  these  colours  may  be  grouped  as  follows : — 

(A.)  Violet  Aniline  Colours. 

Methyl  Violet. — Dissolve  2 grams  in  too  cc.  and  filter.  This 
gives  a 2 per  cent,  solution,  which  may  be  diluted  as  required.  A 
o.  I per  cent,  solution  is,  in  many  cases,  sufficient.  It  does  best  for 
porous  textures.  Sections  are  left  to  stain  in  it  for  several  hours, 
washed  with  water,  and  then  with  alcohol,  until  no  more  colour 
comes  away,  and  mounted  in  balsam.  It  stains  intercellular  sub- 
stances but  slightly,  while  cells,  and  especially  their  nuclei,  are 
stained  by  it. 

Gentian  Violet  always  answers  very  well  for  staining  cell-nuclei, 
but  it  seems  to  be  better  for  hardened  preparations  than  the 
previous  dye.  ' 

Dahli  a is  used  in  the  same  way  as  the  preceding. 

(B.)  Blue  (and  Purple)  Aniline  Colours. 

Aniline  Blue. — Make  a i per  cent,  watery  solution,  adding  a few 
drops  of  absolute  alcohol.  This  is  useful  for  the  glands  of  the 
stomach  and  for  a double  stain  with  safranin. 

Methylene  Blue. — Make  a saturated  watery  solution.  Rectified 
spirit  may  be  added  to  make  it  keep.  It  is  not  very  largely  used 
8 


74 


PRACTICAL  HISTOLOGY. 


in  bacteriology,  e.g.^  for  the  tubercle  bacillus,  but  largely  for  double- 
staining  of  tissues,  in  contrast  to  red.  It  stains  axis  cylinders  of 
nerve  fibres.  (See  Nervous  System  and  Epithelial  Cement.) 

Sections  to  be  mounted  in  balsam  are  best  clarified  by  cedar-oil. 

Better  results  are  obtained  by  using  a very  dilute  solution  made 
as  follows  (Garbini)  : — Add  lo  drops  of  a saturated  alcoholic 
solution  to  loo  cc.  of  a solution  of  caustic  potash  (i  in  10,000). 
Leave  the  sections  to  stain  (12-24  hours).  Leave  them  in  absolute 
alcohol  (6-8  hours),  and  then  in  oil  of  cloves  (2-4  hours) — xylol — 
xylol-balsam. 

Spiller’s  Purple. — Use  Spider’s  purple  No.  i.  Kub  up  2 grams 
in  a glass  mortar  with  10  cc.  of  alcohol,  and  add  100  cc.  of  distilled 
water.  It  is  used  as  a double  stain,  and  for  staining  the  fibrin  in 
coagulated  blood.  The  stain  requires  to  be  pretty  deep,  as  it  is 
washed  out  by  alcohol.  If  a section  stained  by  it  is  to  be  mounted 
in  balsam,  use  cedar-oil  to  clarify  it. 

(C.)  Green  Aniline  Colours. — Amongst  these  are  iodine  green^ 
methyl  green  (i  per  cent.),  aniline  green^  and  aldehyde  green. 

Iodine-Green  is  used  as  a 5 per  cent,  filtered  watery  solution. 
It  stains  nuclei  and  developing  cartilage  green,  and  makes  a good 
contrast  stain.  It  is  not  very  readily  extracted  by  spirit,  and  does 
not  soon  fade. 

Methyl-Green. — This  is  a nuclear  stain.  Make  a i per  cent, 
solution  in  distilled  water,  and  add  25  cc.  of  absolute  alcohol. 
Belgian  observers,  more  particularly  Carnoy,  use  this  for  fresh  tissues 
in  the  following  manner : — 

Methyl-green  .....  i gram. 

Glacial  acetic  acid  . . . . i cc. 

Distilled  water  . . . . . 100  ,, 

Add  a few  drops  of  this  liquid  to  a watch-glassful  of  an  indifferent 
fluid,  e.g..^  normal  saline. 

The  others  will  be  referred  to  in  the  text. 

(D.)  Red  Aniline  Colours. — They  are  very  numerous. 

Rosaniline  Acetate,  Sulphate,  and  Hydrochlorate  (Magenta). — 
The  term  fiichsin  is  sometimes  applied  to  the  one,  sometimes  to 
the  other,  but  the  acetate  is  more  soluble  in  water.  They  give  a 
rather  diffuse  stain,  but  are  useful  for  nuclear  staining,  elastic  fibres, 
and  blood-corpuscles. 

Rub  up  a little  (i  gram)  in  a glass  mortar  with  rectified  spirit 
(20  cc.).  After  solution  add  20  cc.  of  distilled  water. 

Magenta  for  Blood-Corpuscles. — Dissolve  .1  gram  of  magenta 
in  5 cc.  of  rectified  spirit  and  15  cc.  water,  and  add  20  cc.  glycerine. 

Acid  Fuchsin  is  a specific  colouring-matter  for  the  nervous 
system.  It  was  introduced  by  Weigert,  but  it  has  been  largely 
displaced  by  Weigert’s  hsematoxylin  copper  stain. 


STAINING  REAGENTS. 


75 


Safranin  is  specially  used  as  a nuclear  stain,  and  very  largely  for 
the  study  of  mitosis.  It  is  specially  useful  for  tissues  hardened  in 
Flemming’s  mixture. 

Safranin  ...  . . i gram. 

Absolute  alcohol  ....  loo  cc. 

Water  ......  200  ,, 

It  may  also  he  used  as  a much  stronger  alcoholic  solution,  5 per 
cent,  in  70  per  cent,  alcohol.  There  are  several  varieties  of  this 
dye,  and  some  of  them  are  of  little  value  as  dyes.  Therefore  it  is 
im})ortant  to  obtain  a good  sample.  That  sold  as  safranin-0  can 
usually  be  relied  upon,  and  is  to  be  obtained  from  Dr.  George 
Griibler,  Leipzig. 

In  using  this  dye,  the  sections  are,  as  a rule,  left  for  several 
hours  in  the  solution — even  twenty-four  hours  or  longer — and  are 
then  placed  in  ordinary  alcohol  or  acid  alcohol  (p.  65) — containing 
.5  per  cent,  hydrochloric  acid — to  remove  the  surplus  stain.  If  this 
be  properly  done,  the  nuclei — the  chromatin  of  the  nuclei  only — are 
stained.  Sections  may  be  mounted  in  balsam. 

Application. 

(1.)  Stain  sections  in  i per  cent,  watery  solution  of  safranin 
(1-24  hours). 

(2.)  Wash  rapidly  in  water. 

(3.)  Wash  in  absolute  alcohol  or  acid  alcohol. 

(4.)  Absolute  alcohol,  oil,  balsam. 

Sometimes  it  is  useful  to  use  it  in  aniline-water,  after  Ehrlich’s 
method  (p.  73). 

As  shown  by  Martinotti,  it  colours  black  or  dark-purple  elastic 
fibres  hardened  in  0.2  per  cent,  chromic  acid  (Lesson  X.). 

(E.)  Brown  Aniline  Colours. 

Bismarck  Brown  or  Phenylene  Brown.— This  is  but  slightly 
soluble  in  water,  but  it  forms  a good  ground-stain  in  contrast  to 
hsematoxylin,  and  is  useful  for  staining  plasma  cells  and  the  cells  of 
the  cerebrum.  It  preserves  its  colour  when  mounted  in  Tarrant’s 
solution  or  balsam. 

((/.)  Boil  Bismarck  brown  with  100  of  water  ( = 3-4  per  cent.). 
Filter  and  add  one-third  its  volume  of  absolute  alcohol. 

(b.)  Or  use  a concentrated  alcoholic  solution  in  40  per  cent, 
alcohol  ( = 2-2 1 per  cent.). 

Application. 

(1.)  Stain  (5  minutes). 

(2.)  Wash  out  in  strong  spirit. 

(3.)  Alcohol,  oil,  balsam. 

It  does  not  tend  to  over-stain.  It  is  a nuclear  stain,  and  the  nuclei 


76 


PRACTICAL  HISTOLOGY. 


arc  brown,  and  the  protoplasm  light  brown.  Such  preparations  are 
well  adapted  for  photographic  reproductions.  Some  use  a solution 
in  70  per  cent,  alcohol. 

Vesuvin  is  much  more  soluble,  and  is  used  in  the  same  way  as 
Bismarck  brown. 

(F.)  Other  Aniline  Dyes. 

Aniline  Blue-Black. — This  has  a remarkable  power  of  staining 
nerve-cells,  as  shown  by  Sankey.  It  is  best  adapted  for  staining 
fresh  nerve-tissues,  although  it  is  also  used  for  staining  in  bulk. 
It  has  little  power  of  diffusing  through  them.  (Lessons  on  Central 
Nervous  System.) 


Table  shoicing  some  of  the  Aniline  Dyes  in  most  Common  Use^ 
arranged  according  to  their  Colour. 


Red. 

Green. 

Blue. 

Yellow. 

Violet. 

Brown  and 
Black. 

Safraiiin. 
' Eosin. 
Fuchsin. 
Ponceau 
RR. 

Methyl- 

green. 

Iodine 

green. 

Aldehyde 

green. 

Methylene 

blue. 

Aniline 

blue. 

Quinoline. 

Orange. 

Methyl- 

violet. 

Violet 

BBBBB. 

Dahlia. 

Bismarck 

brown. 

Nigrosin. 

Aniline 

blue-black. 

E.  Metallic  Substances. 

1.  Nitrate  of  Silver. — This  substance  possesses  the  property  of 
forming  a compound  with  intercellular  substance,  which  darkens, 
and  becomes  brown  or  black,  on  exposure  to  light.  It  is  unequalled 
for  the  study  of  the  cement  substance  of  epithelium  and  endo- 
thelium, and  for  the  cell-spaces  of  the  cornea,  and  connective  tissue 
generally.  The  tissue,  however,  must  be  fresh — the  fresher  the 
better.  Use  a glass  or  horn  rod  to  manipulate  the  tissue  in  the 
fluid,  not  metallic  instruments. 

Make  a i per  cent,  solution,  i.e.^  i gram  of  silver  nitrate  is 
dissolved  in  100  cc.  • of  distilled  water.  This  is  kept  as  a stock. 
A ^ or  I per  cent,  solution  is  the  strength  usually  employed. 

If  it  be  desired  to  “ silver  a part  of  the  omentum  or  mesentery, 
this  membrane  should  be  pinned  out  with  hedgehog  spines,  without 
being  stretched,  on  a piece  of  flat  cork  with  a large  hole  in  it,  so 
that  the  solution  can  get  to  both  sides  of  the  membrane.  A very 
convenient  plan  is  to  pass  the  membrane  over  a porcelain  or  ebonite 
ring,  and  fix  it  with  another  ring  in  the  manner  in  which  a skin  is 
fitted  on  a drum  (figs.  45  and  46). 

Lave  the  membrane  gently  in  distilled  water  to  remove  any 


STAINING  REAGENTS. 


77 


chlorides,  and  place  it  in  a dilute  solution  of  the  reagent  {\-\-\  per 
cent.).  The  tissue  soon — 5-10  minutes — becomes  white,  and  as 
soon  as  it  looks  grayish  remove  it  and  wash  it  in  distilled  water. 
Place  it  in  ordinary  water,  and  expose  it  to  good  daylight,  when  it 
rapidly  becomes  brown.  It  can  then  be  preserved  in  spirit  until  it 
is  required. 

A membrane  may  be  stained  in  situ^  e.g.^  the  central  tendon  of 
the  diaphragm  of  a rabbit,  or  the  mesentery,  e.g.^  of 
the  abdomen,  irrigate 
the  membrane  by  allow- 
ing distilled  water  to 
fall  on  it  from  a pipette. 

This  removes  all  sub- 
stances that  might  com- 
bine with  the  silver  and 
give  rise  to  illusive  ap- 
pearances. Drop  on  the 
silver  solution  by  means 
of  a pipette.  Treat  the 
membrane  as  in  the 

previous  case.  I’ig.  45.— a.  Vulcanite  rings  used  for  stretching  a mem- 

2.  Negative  Method  is  to  be  silvered  ; B.  Conical  rings,  the 

(Recldinghausmy—HMs  ^ 

is  the  usual  method  by  which  tlie  intercellular  cement  substance  is 
stained  black — a weak  solution  being  used — as  for  the  study  of 
endothelium.  The  membrane 
ought  to  be  kept  stretched, 
e.g.,  over  the  mouth  of  a 
porcelain  capsule. 

The  following  modification 
is  recommended  by  Thanhoffer, 
and  it  works  well.  After  the 
membrane  is  exposed  to  the 
action  of  silver  nitrate,  it  is 


.—Section  of  A.  a.  Inner,  and  h.  Outer 
ring ; M.  Stretched  membrane. 


washed  with  a 2 per  cent,  solution  of  acetic  acid. 

3.  Positive  Method  (His)  is  specially  designed  for  showing  lacun<e 
and  lymphatic  cavities,  e.g.^  the  lymphatics  of  the  skin  and  cornea. 
Place  the  tissue  for  several  hours  in  i per  cent.  AgNOg  in  the  dark, 
wash  in  water,  transfer  in  dark  to  3.5  solution  of  sodic  chloride! 

ash  in  water,  expose  to  light.  The  spaces  are  found  filled 
with  black  granules.  Examine  in  glycerine. 

Sometimes  silver  nitrate  is  used  in  a solid  form,  e.q.^  for  the 
cornea.  This  will  be  referred  to  afterwards  in  treating  of  the  cell- 
spaces  of  the  cornea  itself.  (Lesson  on  Eye.) 

Silver  preparations  show  the  cement  substance  of  epithelium 


78 


PRACTICAL  HISTOLOGY. 


stained  black  as  “ silver  lines/’  and  they  may  be  mounted  in 
glycerine  or  balsam,  either  unstained  or  after  staining  with  logwood, 
picro-carmine,  or  other  dye  as  desired. 

In  order  to  stain  the  lining  endothelium  of  the  vascular  system, 
a solution  of  silver  nitrate  is  used.  The  special  precautions 
required  are  referred  to  in  the  text.  (Lesson  on  Blood-Yessels.) 

4.  Golgi’s  Method. — In  this  method  parts  of  the  central  nervous 
system  hardened  in  potassic  bichromate  are  treated  for  many  days 
with  silver  nitrate  or  mercuric  chloride  to  demonstrate  the  processes 
of  nerve-cells.  (Lesson  on  Nervous  System.) 

5.  Nitrate  of  Silver  and  Osmlc  Acid  {Golgi), — This  is  specially 
useful  for  the  nervous  system.  Place  a fresh  nerve  of  a rabbit  just 
killed  in 

Potassic  bichromate  (2  per  cent.)  . . lo  parts 

Osniic  acid  (1  per  cent.)  . . . 2 ,, 

for  an  hour ; tease  the  nerve,  and  let  it  remain  in  the  mixture  for 
another  hour.  Transfer  for  8 hours  to  . 5 per  cent,  silver  nitrate  and 
then  to  alcohol. 

B.  Gold  Chloride. 

Gold  Chloride. — This  substance  has  rendered  particular  service, 
especially  in  connection  with  the  terminations  of  nerves.  It  is  used 
as  \-2  per  cent,  watery  solution.  Various  methods  are  employed, 
according  to  the  end  desired. 

1.  Acetic  Acid  Method. — Place  a small  piece  of  perfectly  fresh 
tissue,  2 mm.  cubes,  e.g.,  hyaline  cartilage  or  a small  cornea,  in  -|-per 
cent,  solution  in  a glass  thimble  for  half-an-hour,  keeping  it  in  the 
dark  all  the  time.  These  small  glass  thimbles  are  particularly  use- 
ful, and  are  better  to  be  somewhat  broader  relatively  than  those 
shown  in  fig.  26.  The  tissue  will  become  yellow ; wash  it 
thoroughly  in  distilled  water,  and  expose  it  to  bright  daylight  in 
distilled  water  slightly  acidulated  with  acetic  acid.  In  a day  or 
two  it  will  become  of  a purplish  or  violet-brown  colour.  Sections 
can  then  be  made  and  mounted  in  glycerine. 

2.  Loewit’s  Method. — To  one  part  of  formic  acid  (sp.  gr.  i.i6) 
add  two  parts  of  distilled  water.  Place  small  pieces  of  the  fresh  tissue 
(1-2  mm.  in  thickness),  e.g.,  tendon  from  a rat’s  tail,  in  this  mixture 
for  |-i  minute,  until  they  become  somewhat  transparent;  transfer 
them  to  a glass  thimble  or  watch-glass  containing  i per  cent,  gold 
chloride  for  1:5-20  minutes,  i.e.,  until  they  have  become  yellow 
throughout,  jluring  this  process,  the  tissue  should  be  exposed  to 
light  as  little/as  possible.  Place  the  tissue  in  formic  acid  (i  13), 
and  keep  it  in  the  dark  for  twenty-four  hours.  Afterwards  place  it 
for  twenty-four  hours  in  pure  formic  acid,  and  keep  it  also  in  the 


STAINING  REAGENTS. 


79 


dark.  Wash  it  thoroughly  with  water,  and  mount  in  glycerine  or 
balsam. 

3.  Ranvier’s  Lemon-Juice  Method. — The  fresh  tissue  is  placed 
for  5-10  minutes  in  the  freshly  expressed  and  liltered  juice  of  a 
lemon,  until  it  becomes  transparent.  Rapidly  wash  it  in  distilled 
water,  transfer  it  to  i per  cent,  gold  chloride  solution  for  from  ten 
minutes  to  one  hour ; the  time  depends  on  the  tissue  under  investi- 
gation. Wash  with  water  and  place  the  tissues  in  50  cc.  of  water 
containing  two  drops  of  acetic  acid,  and  expose  them  to  light,  when 
reduction  takes  place.  Or  the  tissue  may  be  placed  in  formic  acid 
(i  : 3)  after  being  treated  with  lemon-juice  and  gold  chloride,  and 
kept  in  the  dark  for  twenty-four  hours.  The  latter  plan  is  in  many 
cases  to  he  preferred,  especially  where  the  retention  of  the  super- 
ficial epithelium  is  not  desired. 

4.  Boiled  Gold  Chloride. — For  some  purposes,  especially  for 
studying  the  terminations  of  the  nerves  in  sensory  surfaces,  this 
method  of  Ran  vie  r has  yielded  me  the  best  results. 

Make  as  required — fresh — a mixture  of  four  parts  of  gold 
chloride  (i  per  cent.)  and  one  part  of  formic  acid.  Boil  the 
mixture  and  let  it  cool.  Place  the  fresh  tissues  (small  pieces)  in  it 
for  ten  minutes  to  one  hour.  Wash  in  water,  and  ])lace  in  formic 
acid  (1:4  water),  and  keep  in  darkness,  where  the  reduction 
takes  place. 

5.  Rapid  Reduction  of  Gold  Chloride. — A tissue  may  be  left  in 
gold  chloride  (i  per  cent.)  for  half-an-hour  or  more,  and  then  trans- 
ferred to  a strong  solution  of  tartaric  acid  and  heated  to  45”  or  50° 
C.,  when  it  rapidly  becomes  of  a purplish-brown  colour,  usually  in 
the  course  of  a quarter  of  an  hour. 

Although  it  has  been  stated  that  for  gold  chloride  preparations 
the  tissues  should  be  fresh,  Drasch,  in  his  researches  on  the  nerves 
of  the  intestine  and  those  of  the  circumvallate  papillae,  points  out 
that  he  obtained  the  best  results  with  tissues  twenty-four  hours 
after  death ; the  tissues,  however,  must  have  been  kept  cool.  * Any 
one  who  has  had  the  privilege  of  studying  the  preparations  of 
Drasch  cannot  hut  have  been  impressed  with  the  beauty  of  speci- 
mens prepared  by  his  method. 

6.  Gold  Chloride  and  Chromic  Acid  {Kolossow). — Place  the 

tissues  for  2-3  hours,  according  to  their  size,  in  i per  cent,  gold 
chloride  acidulated  with  hydrochloric  acid  (i-ioo).  Wash  the 
tissues  with  water,  and  keep  them  in  the  dark  in  chromic  acid 
(5V  cent.)  for  2-3  days.  Wash  out  the  chromic  acid 

thoroughly. 

7.  Method  of  Ciaccio  is  good  for  the  termination  of  nerves  in 
cornea  and  muscles.  Place  a small  piece  of  tissue,  not  more  than  2 
cubic  mm.  in  size,  in  fresh  juice  of  lemon  (5  minutes) : wash ; place  in 


8o 


PRACTICAL  HISTOLOGY. 


I per  cent,  solution  of  chloride  of  gold  and  cadmium  (30-60  minutes) 
in  the  dark.  Wash  ; then  in  i per  cent,  formic  acid  (24  hours) 
in  the  dark,  and  then  for  12  hours  in  sunlight.  Finally  for  24  hours 
in  pure  formic  acid.  Wash.  Tease  and  mount  in  glycerine. 

F.  Double,  Treble,  or  Multiple  Stainings. 

It  is  possible  to  stain  a section  so  that  the  several  parts  of  it  may 
be  differently  stained.  This  may  be  done  either  by  staining  succes- 
sively with  different  stains,  or  by  mixing  the  dyes  in  one  fluid,  and 
staining  the  section  with  the  mixture,  whereby  one  part  takes  up 
one  of  the  dyes  and  another  part  one  of  the  other  dyes.  Thus  one 
gets  an  elective  and  differential  stain.  It  is  possible  thus  to  combine 
a nuclear  stain  with  one  which  stains  only  the  protoplasm  of  the 
cells,  such  as  eosin  or  orange. 

Amongst  double  stains  are  the  following  : — 

1.  Picro-carmine  (p.  66),  one  of  the  most  valuable  we  possess, 
and  which  we  owe  to  Kanvier ; and  Picro-litho-carmine.  If  the 
section  is  to  be  mounted  in  glycerine  or  FarranFs  solution,  do  not 
ivash  it  in  water.  If  it  is  to  be  mounted  in  balsam,  the  alcohol  in 
which  it  is  washed  should  contain  picric  acid — the  same  result  is 
obtained  by  using  clove-oil  with  picric  acid  dissolved  in  it — other- 
wise only  a carmine  stain  is  obtained. 

2.  Carmine  and  Aniline  Blue. — Stain  a section  in  borax-carmine, 
and  then  in  very  dilute  aniline  blue,  specially  useful  for  the  glands 
of  the  fundus  of  the  stomach.  In  other  tissues  the  nuclei  are  red 
and  the  perinuclear  parts  blue. 

3.  Haematoxylin  and  Eosin. — Haematoxylin  is  a nuclear  stain, — 
the  sections  are  first  stained  in  it,  and  then  in  eosin,  which  stains 
the  general  protoplasm  of  the  cell.  (See  also  p.  72.) 

4.  Aniline-blue  and  Safranin  (Garbini). — The  section  is  trans- 
ferred from  water. 

Manipulation. 

Aniline-blue  sol.  (.5  per  cent.),  2-4  minutes. 

Wash  in  water. 

Lithium  carbonate  (.5  per  cent.),  a few  minutes. 

Hydrochloric  acid  (.5  per  cent.),  a transparent  blue  colour 
is  produced. 

Wash  in  water. 

Safranin  (i  per  cent.),  10  minutes. 

Dehydrate  in  met  hylic  alcohol. 

Clarify  in  oil  of  cloves  (2  parts)  and  cedar-oil  (i  part). 

Xylol-balsam. 

This  is  especially  useful  for  some  of  the  salivary  glands — thus  in 
the  sub-maxillary  one  set  of  cells  is  red,  the  other  blue ; in  the 
stomach  the  parietal  cells  are  red  and  the  inner  blue  ; the  epithelial 


STAINING  REAGENTS. 


8l 


cells  of  the  villi  are  blue,  the  goblet  cells  reddish ; in  a hair-follicle 
the  sheath  of  Henle  is  an  intense  red,  and  the  sheath  of  Huxley 
blue. 

5.  Ehrlich-Biondi-Heidenhain  Stain. 

Saturated  watery  solution  of  orange  . . loo  cc. 

,,  ,,  ,,  acid  fuclisin  . 20  ,, 

,,  ,,  ,,  methyl -green  . 50  ,, 

To  get  complete  saturation  it  is  necessary  to  have  an  excess  of 
the  crystals  for  several  days.  Each  fluid  is  saturated  separately. 
Before  use,  the  solution  is  diluted  in  the  proportion  of  i in  100  with 
water,  and  then  on  the  addition  of  acetic  acid  must  be  bright  red. 
It  is  better  to  obtain  the  mixture  from  Dr  Griibler. 

Application. 

(1.)  Harden  the  organ  in  corrosive  sublimate. 

(2.)  Stain  sections  in  the  dilute  solution  (12-24  flours). 

(3.)  Wasfl  quickly  in  90  per  cent,  alcoflol. 

(4.)  Deflydrate  in  absolute  alcohol. 

(5.)  Xylol-balsam. 

It  is  especially  useful  for  sections  containing  many  leucocytes, 
and  is  best  used  for  paraffin  sections  fixed  on  a slide.  Red  blood- 
corpuscles  are  stained  red,  resting  nuclei  blue,  mitotic  figures  and 
nuclei  of  leucocytes  green-violet. 

G-eneral  Remarks  on  Staining. — Filter  the  staining  fluid. 
When  possible,  use  a tveak  solution  of  the  dye,  and  thus  let  the 
sections  stain  slotoly  in  a fairly  large  amount  of  the  fluid.  Place  a 
piece  of  blotting-paper  on  the  inside  of  a large  watch-glass,  pour 
some  of  the  diluted  stain  into  the  watch-glass,  and  place  the  sections 
in  it.  The  sections  should  lie  as  flat  as  possible,  and  not  overlap 
each  other.  The  sections  may  be  moved  gently  in  the  fluid  by 
means  of  a needle.  Cover  the  watch-glass  with  another  glass  of 
the  same  size,  or  set  it  aside  in  a moist  chamber,  e.g.^  on  a plate 
covered  by  a bell-jar,  with  a piece  of  moistened  blotting-paper 
attached  to  the  inside  of  the  jar  (fig.  47).  After  staining,  the 
sections  are  to  be  carefully  washed  in  distilled  water  to  remove  any 
trace  of  surplus  dye  (except  in  the  case  of  picro-(iarmine).  Sections 
stained  with  hsematoxylin  should  be  washed  for  a long  time  in 
water  before  they  are  mounted  in  glycerine  or  Tarrant’s  solution. 

Be  careful  not  to  over-stain  the  tissue,  except  in  those  cases 
where  the  excess  can  be  again  removed,  e.g.^  with  the  aniline 
dyes  by  means  of  alcohol  or  acid  alcohol. 

All  acids  should  be  removed  from  the  sections  before  they  are 
placed  in  the  dye. 

It  is  convenient  on  many  occasions  that  the  student  should 
rapidly  stain  his  sections  on  a slide,  but  he  should  also  be  taught 

9 F 


82 


PRACTICAL  HISTOLOGY. 


to  practise  the  slower  method  of  staining  sections  in  very  dilute 
solutions  of  a dye. 

There  is  one  method  of  staining  sections  which  may  be  profitably 
impressed  upon  the  student,  viz.,  that  so  strongly  insisted  upon  by 

Ranvier.  Suppose  any 
delicate  object— isolated 
epithelial  or  other  cells 
— to  be  mounted  in  a 
watery  medium ; a drop 
of  a solution  of  picro- 
carmine  is  placed  at  one 
side  of  the  cover-glass. 
As  the  fluid  evaporates 
at  one  side  of  the  cover- 
glass  the  picro-carmine 
slowly  diflPuses  under  the 
cover-glass  and  stains 
the  preparation.  Ex- 
posed to  the  air,  the 
preparation  would  soon 
become  dry.  This  must 
be  corrected.  This  is 
best  done  by  placing 
the  slides  to  be  stained 
in  this  ^ way  on  a stage 
with  several  shelves 
(fig.  47),  the  whole  being  placed  on  a plate  moistened  with  a few 
drops  of  water  and  covered  by  a bell-jar.  This  forms  a moist 
chamber. 

After  the  cells  are  stained,  glycerine  may  then  be  applied  at  the 
side  of  the  cover-glass,  with  the  same  protective  precautions,  so 
that  the  preparation  can  be  Anally  mounted  and  preserved  in 
glycerine. 


XIII.— CLEABINQ  OR  CLARIFYING  REAGENTS. 

Glycerine,  Farrant’s  Solution,  and  Glycerine  Jelly. — When 
any  one  of  these  reagents  is  used  for  mounting  preparations,  no 
other  clarifying  substance  is  used. 

Balsam  Preparations. — When  a preparation  is  to  be  mounted 
in  balsam,  be  it  Canada  balsam  or  dammar,  some  clarifying  reagent 
has  to  be  added  to  the  preparation  before  the  balsam  is  applied. 
Before  applying  the  balsam  the  tissues  must  have  been  rendered 
transparent. 


CLEARING  OR  CLARIFYING  REAGENTS. 


83 


The  following  substances  are  most  commonly  used  : — Oil  of 
cloves,  a mixture  of  creosote  and  turpentine,  turpentine,  creosote, 
xylol,  cedar-oil,  bergamot  oil,  lavender  oil,  origanum  oil,  &c. 

Oil  of  Cloves  has  this  advantage,  that  it  clarifies  rapidly  and 
does  not  evaporate,  so  that  sections  may  be  left  exposed  to  the  air  in 
it  for  some  time.  It  renders  the  sections  very  hard.  It,  however, 
is  not  so  satisfactory  for  aniline  dye  specimens,  as  it  is  apt  to  abstract 
their  colour.  Moreover,  it  becomes  yellow  with  age. 

Creosote  is  specially  useful  for  preparations  which  one  does  not 
desire  to  harden  in  alcohol ; do  not  use  metallic  instruments. 

Creosote  and  Turpentine. — When  the  fluids  are  mixed  a cloudi- 
ness appears,  but  this  disappears  on  keeping.  It  is  much  cheaper 
than  clove-oil,  but  it  rapidly  evaporates  (one  part  creosote  to  four  of 
turpentine). 

Xylol  is  perhaps  the  best,  and  is  specially  useful  with  aniline 
dyes.  In  these  cases  the  balsam — Canada  or  dammar — should  also 
be  dissolved  in  xylol. 

Cedar-Wood  Oil  clarifies  very  slowly.  It  does  not,  however, 
abstract  the  aniline  dyes,  and  is  used  for  special  purposes,  as  indi- 
cated in  the  context. 

Origanum  Oil  is  used  for  clarifying  sections  embedded  in 
celloidin. 

Xylol- Aniline  Oil. — Equal  parts  of  xylol  and  aniline  are  used  for 
clarifying  sections  under  certain  conditions  without  the  previous  use 
of  alcohol.  (Weigert’s  method.  Lesson  III.) 

Carbolic  Acid  and  Xylol. — A mixture  of  i part  of  carbolic  acid 
and  3 of  xylol  is  used  to  clarify  celloidin  sections  (p.  47).  The 
section  can  be  taken  from  70  per  cent,  alcohol,  and  does  not  require 
to  be  further  dehydrated.  To  remove  the  water  from  the  mixture, 
keep  in  the  bottom  of  the  bottle  containing  it  a thick  layer  of 
previously-heated  copper  sulphate. 

General  Remarks. — Although  several  essential  oils  are  used  for 
clarifying  purposes,  it  is  not  immaterial  which  one  is  used.  Thus 
clove-oil  may  be  used  for  clarifying  sections  stained  with  animal 
or  vegetable  dyes  (carmine),  while  it  is  inapplicable  for  aniline 
staining,  as  it  dissolves  aniline  dyes. 

In  many  cases  the  result  may  be  obtained  more  gradually  by 
using  a mixture  of  half  alcohol  and  half  essential  oil. 

Moreover,  clove-oil  dissolves  celloidin,  so  that  it  cannot  be  used 
when  the  section  fixed  on  the  slide  contains  either  celloidin  or 
collodion.  Oil  of  bergamot  does  not  dissolve  celloidin. 


84 


PRACTICAL  HISTOLOGY. 


In  alcohol  (70  p.c.). 
Carmine. 

Acid  alcohol. 
Alcohol  (70  p.c.). 
Alcohol  (90  p.c.). 
Alcohol  (absolute). 
Oil  of  cloves. 
Balsam. 


Scheme  for  Staining  (Garbini). 

Object. 


Embedding. 


Sections  in  celloidin. 


Sections  in  paraffin. 
Turpentine. 


Absolute  alcohol. 


Alcohol  (70  p.c.). 


Carmine. 

! 

Acid  alcohol. 
Water. 


Hsematoxylin. 
Acid  alcohol. 
Water. 

Carb.  of  Lithia. 


Aniline  dye. 


Absolute  alcohol. 

Oil  of  cloves  or  bergamot  oil. 
Xylol. 

Balsam. 


MOUNTING  FLUIDS,  AND  METHODS. 


35 


XIV.— MOUNTING  FLUIDS,  AND  METHODS. 

The  fluid  chosen  will  depend  on  the  nature  of  the  tissue  and 
other  circumstances. 

1.  If  a section  is  to  be  mounted  direct  from  water,  glycerine, 
•Farrant’s  solution,  or  glycerine  jelly  may  be  used. 

2.  If  a section  is  to  be  mounted  in  balsam,  it  must  have  every 
trace  of  water  removed  by  alcohol,  and  the  alcohol  must  be  dis- 
placed by  one  of  the  clarifying  reagents — xylol,  clove-oil,  &c. — 
already  mentioned. 

Glycerine. — Pure  glycerine  is  only  used  for  such  tissues  as 
have  been  previously  hardened.  In  the  case  of  tissues — delicate 
tissues  which  have  not  been  previously  hardened  or  fixed — the 
direct  application  of  pure  glycerine  would  injure  them.  In  this 
case,  the  best  way  is  to  mount  the  object  in  normal  saline,  and  at 
one  edge  of  the  cover-glass  to  place  a drop  of  a mixture  of  equal 
parts  of  glycerine  and  water.  Put  the  preparation  in  a plate 
covered  by  a bell-jar — an  extempore  moist  chamber.  The  glycerine 
slowly  penetrates  as  the  water  evaporates. 

Some  tissues  are  rendered  too  transparent  by  glycerine,  and, 
moreover,  it  is  very  difficult  to  seal  up  and  keep  tight  glycerine 
preparations. 

Glycerine  and  Formic  Acid. — This  is  sometimes  used,  especially 
for  picro-carmine  preparations.  It  is  made  by  adding  formic  acid 
to  dilute  glycerine  (i  per  cent.). 

Farrant’s  Solution. — This  is  for  many  preparations  far  more 
serviceable  than  glycerine,  as  it  does  not  render  some  tissues  so 
transparent  as  glycerine,  and  the  preparations  can  be  easily  sealed 
up  or  ‘‘  ringed.’’ 

Preparation  {Hamilton's  receipt). — Make  a saturated  solution 
of  arsenious  acid  in  water  by  boiling.  After  standing  for  twenty- 
four  hours  filter.  Take  equal  quantities  of  water,  glycerine,  and 
arsenious  water,  and  to  the  mixture  add  picked  gum-arabic.  Let 
the  latter  dissolve  until  a thick  syrupy  fluid  is  obtained,  which  takes 
about  a week  at  an  ordinary  temperature;,  but  it  must  be  stirred 
frequently.  Filter  slowly  through  filter-paper,  which  must  be  fre- 
quently changed. 

Glycerine  Jelly. — Melt  it  in  hot  water,  place  a drop  on  the 
section,  apply  a cover-glass,  and  gently  press  it  down.  It  gelatinises 
in  a few  minutes. 

Canada  Balsam. — Place  some  Canada  balsam  in  a capsule  or 
wide-mouthed  bottle  near  a fire  or  in  a warm  chamber  (65°  C.) 
until  it  becomes  hard.  Let  it  cool.  This  dry  balsam  is  to  be  dis- 
solved in  some  medium.  8ome  use  chloroform,  others  benzol, 
others  a mixture  of  both,  or  turpentine  as  a solvent.  In  any  case. 


86 


PRACTICAL  HISTOLOGY. 


Fig,  48.— Capped  Bottle  for 
Balsam. 


the  solvent  is  added  until  a fairly  thin  fluid  is  obtained.  Perhaps 
the  best  solvent  of  all  is  xylol.  It  requires  nearly  twice  its  volume 
of  xylol.  Filter  through  paper.  The  balsam  should  be  kept  in  a 
‘‘capped’^  bottle  (fig.  48)  instead  of  a 
stoppered  one.  If  it  gets  too  thick,  add  a 
little  xylol. 

Dammar  Lac  {Klein), 

Gum  dammar  . . . i J oz. 

Gum  mastic  . . . ^ 

Turpentine  . . . 2 , , 

Chloroform  . . . 2 ,, 

Dissolve  the  dammar  in  the  turpentine, 
and  filter ; the  mastic  in  the  chloroform, 
and  filter.  Mix  the  two  solutions  and 
filter  again. 

There  must  be  no  moisture  in  the 
bottles,  and  the  mixture  must  be  kept  in 
‘ capped  bottles,  else  the  chloroform  will 
evaporate. 

Xylol-Balsam. — Dry  ordinary  Canada 
balsam  in  a sand-bath,  to  drive  off  all  the 
moisture,  and  until  it  becomes  vitreous. 

If  it  be  spread  out  in  a thin  layer  in  a tin 
vessel,  this  is  usually  accomplished  in  two  hours  or  so,  but  the 
balsam  must  not  be  overheated  or  change  its  colour  and  become 
brown.  Dissolve  the  dried  balsam  in  an  equal  volume  of  xylol. 
Perhaps  this  is  the  best  form  of  balsam  to  use. 

Balsam,  when  prepared,  should  be  kept  in  a glass  bottle  with  a 
ground-glass  cap. 

To  Place  a Section  on  a Slide. — By  far  the  most  convenient 
method  is  to  place  the  section  in  a basin  of  water.  Hold  the  slide 
perpendicularly  by  the  edges  in  the  left  hand,  plunge  the  slide  into 
the  water  until  it  is  about  three-fourths  immersed,  and  with  a 
mounted  needle  pull  the  section  on  to  the  slide,  and  at  the  same 
moment  raise  the  latter  out  of  the  water.  The  section  adheres  to 
the  glass,  and  if  it  be  folded  at  one  end,  dip  this  end  in  the  water, 
when  it  floats  out  quite  flat.  Do  not  attempt  to  spread  out  the 
folds  on  the  slide  by  means  of  a needle. 

Hold  the  slide  vertically  to  allow  the  water  to  drain  off,  and 
remove  with  a rag  or  well-washed  cloth  the  remainder  of  the  water 
close  up  to  the  section. 

It  may  be  stained  on  the  slide.  After  the  staining  is  complete, 
remove  the  surplus  dye  by  means  of  bibulous  paper,  taking  care, 
however,  that  the  section  itself  does  not  adhere  to  the  absorbent 
paper. 


MOUNTING  FLUIDS,  AND  METHODS.  8/ 

If  the  section  is  to  he  mounted  in  glycerine  or  Farranfs  solution^ 
add  a drop  of  either  of  these  reagents  and  apply  a cover-glass. 

If  the  section  is  to  he  mounted  in  halsam,  remove  as  much  as 
possible  of  the  surplus  water  or  dye,  as  the  case  may  be,  and  pour 
methylated  spirit  upon  the  section.  Allow  it  to  remain  on  the 
section  for  a minute  or  so,  and  drain  it  off  at  one  end  of  the  slide. 
Apply  fresh  methylated  spirit  again,  and  finally  absolute  alcohol. 
This  is  done  to  secure  complete  dehydration.  The  frequent  and 
prolonged  application  of  strong  spirit  removes  all  the  water. 

Remove  as  much  of  the  spirit  as  possible,  but  do  not  allow  the 
section  to  dry.  It  is  now  ready  to  be  cleared  up. 

With  a brush  insinuate  a drop  of  the  clarifying  reagent — clove- 
oil  or  xylol — under  one  corner  of  the  section,  and  allow  the  xylol  to 
flow  under  the  whole  of  the  section.  It  will  gradually  diffuse  into 
the  tissue  ; and  if  the  process  be  watched  under  the  microscope 
with  a low  power,  the  section  will  be  seen  to  become  gradually 
more  transparent,  while  the  spirit  will  be  seen  os  fine  globules 
driven  out  into  the  essential  oil.  The  success  of  the  process 
depends  on  complete  removal  of  the  water  by  spirit,  and  the  complete 
removal  of  the  latter  by  the  essential  oil  used  as  the  clarifying  re- 
agent. If  any  opacity  remains,  and  it  looks  milky  or  like  an 
emulsion,  there  has  been  either  water  or  spirit,  or  both,  left  in  the 
section. 

More  of  the  essential  oil  is  placed  on  the  section,  so  that  it  is 
completely  bathed  in  it  and  rendered  quite  clear  by  it.  Pour  off 
the  superfluous  oil,  remove  the  surplus  close  up  to  the  edge  of  the 
section,  add  a drop  of  balsam,  apply  a cover-glass,  and  the  process 
is  complete. 

In  some  cases  it  is  convenient  to  put  the  drop  of  balsam  on  the 
cover-glass,  and  then  to  invert  this  on  the  clarified  preparation. 

In  all  cases  where  it  is  directed  to  mount  in  balsam,  this  process 
must  be  gone  through,  viz. — ^ 

(1.)  Stain  the  section. 

(2.)  Wash  it  in  water. 

(3.)  Treat  the  section  with  strong  alcohol  (96  per  cent.)  to  remove 
water  (3-5  minutes). 

(4.)  Absolute  alcohol  (3-5  minutes). 

(5.)  Clarify  with  an  essential  oil  to  remove  all  the  alcohol. 

(6.)  If  the  section  be  not  on  a slide  already,  place  it  on  a slide  by 
means  of  a lifter.  Remove  surplus  oil  with  blotting-paper. 

(7.)  Add  balsam,  cover  the  section  with  a cover-glass. 

(8.)  If  desired,  the  hardening  of  the  balsam  may  be  hastened  by 
gently  warming  the  preparation  on  a water-bath. 

Sometimes  it  is  not  convenient  to  stain,  dehydrate,  and  clarify  a 
section  on  a slide.  In  this  case  the  sections  are  stained,  dehydrated. 


88 


PRACTICAL  HISTOLOGY. 


and  clarified  in  watch-glasses,  the  sections  being  transferred  from 
one  fluid  to  the  other,  and  finally  to  the  slide  by  means  of  a “ lifter  ’’ 
(P-  3)-  . 

Sometimes  the  one  method  is  adopted,  sometimes  the  other. 

To  Clean  a Microscopic  Preparation. — Any  excess  of  balsam 
round  the  edge  of  a preparation  may  be  moved  with  a cloth  dipped 
in  benzol. 

In  the  case  of  a preparation  mounted  in  glycerine^  any  excess  of 
the  latter  must  be  removed  with  great  care,  otherwise  the  cement 
will  not  adhere  to  the  glass. 

With  preparations  mounted  in  Farranfs  solution,  leave  them  in 
an  airy  dry  place  for  ten  days  or  longer  ; this  gives  the  medium 
time  to  harden  at  the  edges,  and  fixes  the  cover-glass  pretty  firmly 
to  the  slide.  Place  the  slide  in  a basin  of  water,  and  with  a 
cameFs-hair  brush  brush  away  from  the  edge  of  the  cover-glass 
every  trace  of  the  medium.  There  is  no  fear  of  disturbing  the 
cover-glass.  Lave  the  slide  in  fresh  water,  and  then  wipe  it 
thoroughly  dry.  It  is  better  to  wash  a number  of  slides  at  a 
time. 

To  cement  or  “ Ring’'  the  Specimens. 

Balsam  Preparations  need  not  be  touched.  They  keep  perfectly 
without  being  covered  in  by  coating  the  edge  of  the  cover-glass 
with  an  adhesive  and  resistant  cement.  If  it  be  desired  to  cement 
them,  a thin  coating  of  Hollis’s  glue  must  first  be  applied,  and  after 
it  is  dry  the  cement  is  laid  on  as  directed  for  preparations  mounted 
in  Tarrant’s  solution. 

To  Ring  a Slide. — The  slide  should  be  fixed  on  a turntable, 
the  centre  of  the  circular  cover-glass  corresponding  to  the  centre  of 
the  brass  disc  of  the  table.  The  slide  is 
fixed  in  position  by  means  of  two  brass 
clips  (figs.  49,  a,  h,  50). 

For  Farr  ant's  preparations  or  glycerine 
preparations,  lay  on  a ring  of  white  zinc 
cement  with  a goat’s-hair  brush.  The  disc 


Fig.  49.— Turntable  for  Ringing  Slides. 

is  made  to  revolve  with  the  fore-finger  of  the  left  hand,  but  not  too 
quickly,  and  a coating  of  the  cement  is  laid  on  evenly.  The  fore- 
finger is  applied  to  the  smaller  disc  (c). 


Fig.  50. — Showing  how  slide 
is  to  be  centred  on  the 
Turntable. 


INJECTING  BLOOD-VESSELS  AND  GLAND-TUBES.  8q 


The  turntable  should  be  heavy  and  mounted  on  a pin-point 
centre-piece.  The  brushes  must  not  be  too  large,  and  should  be 
washed  immediately  after  use  in  the  same  fluid  as  is  used  to  dis- 
solve the  cement.  Thus,  for  zinc-white  the  brush  is  to  be  washed 
in  benzol  or  xylol,  and  for  gold-size  in  turpentine,  and  for  Tarrant’s 
solution  in  water. 

White  Zinc  Cement. — Dissolve  3 oz.  of  dammar  in  3 oz.  of 
benzol,  and  add  200  grains  of  finely-ground  oxide  of  zinc.  Mix  the 
whole  thoroughly,  and  strain  through  several  folds  of  muslin.  It  is 
perhaps  more  convenient  to  purchase  the  cement. 

Mounting  Block. — It  is  important  that  the  section  be  placed 
in  the  centre  of  the  slide.  As  a guide  for  this  purpose,  cut  a 
piece  of  paper  the  size  of  the  slide,  and  draw  diagonal  lines  from 
corner  to  corner  of  it ; they  will  intersect  in  the  centre.  Or  the 
piece  of  paper  may  be  gummed  by  means  of  Hollis’s  glue  between 
two  slides. 


XV.— INJECTING-  BLOOD-VESSELS  AND 
GLAND-TUBES. 

Transparent  Injection  Masses. — At  the  present  time,  histologists 
use  transparent  injections,  consisting  of  a vehicle — which  may  be 
water,  glycerine,  or  gelatine — and  a colouring  matter.  Most 
commonly  gelatine  is  used  as  a vehicle.  The  colouring  matter  of 
most  red  injections  is  carmine.  In  this  case,  the  secret  is  to  have 
the  mass  as  neutral  as  possible. 

1.  Carter’s  Carmine  Injection. 


Carmine  . . . . . i dr. 

Stroiiir  solution  of  ammonia  . . . 2 fl.  drs. 

Glacial  acetic  acid  . . . .86  mins. 

Solution  of  gelatine  (i  to  6 water)  . . 2 oz. 

Distilled  water  . . . . 


Rub  up  the  carmine  with  a little  water  in  a mortar,  add  the 
remainder  of  the  water,  and  then  add  the  ammonia,  and  stir  until 
the  carmine  - is  dissolved.  Add  the  glacial  acetic  drop  by  drop, 
stirring  thoroughly.  Add  the  gelatine  solution,  and  stir  briskly. 

2.  Banvier’s  Method. — The  following  method  yields  excellent 
results.  Mix  2-5  grms.  of  pure  carmine  with  a little  distilled 
water  in  a stoppered  bottle,  and  add  ammonia  solution,  drop  by 
drop,  until  the  carmine  is  dissolved,  which  occurs  when  the  liquid 
becomes  transparent.  Shake  up  the  liquid  to  get  it  homogeneous. 

Weigh  5 grms.  of  dry  Paris  gelatine  (Coignet’s),  and  place  it  in 
distilled  water  for  one  hour.  At  the  end  of  this  time  it  is  swollen 
up  and  soft.  Remove  it  from  the  water,  v^ash  it  in  water,  and 


90 


PRACTICAL  HISTOLOGY. 


place  it  in  a beaker  in  a water-bath.  When  the  gelatine  is  dissolved 
by  the  water  which  it  has  absorbed,  add  to  it — stirring  vigorously — 
the  solution  of  carmine,  which  yields  an  amrnoniacal  solution  of 
carmine  in  gelatine. 

When  the  carmine  mixture  is  on  the  water-bath  make  a solution 
of — 

Distilled  water  . . . . .2  parts. 

Glacial  acetic  acid  . . . . i part. 

Pour  the  acid  drop  by  drop  into  the  mass,  stirring  thoroughly 
all  the  time  with  a glass  rod.  The  acid  is  to  neutralise  the  excess 
of  ammonia.  This  requires  great  attention.  It  is  by  the  odour 
that  one  recognises  when  the  fluid  is  neutralised.  As  the  acid  is 
added  the  ammoniacal  odour  diminishes,  and  there  is  at  last  a 
faint  acid  odour.  This  is  the  moment  to  stop  adding  the  acid. 
Towards  the  end  of  the  operation  it  is  best  to  dilute  the  acid 
somewhat. 

Filter  the  mass  through  new  flannel. 

3.  Carpaine  Gelatine  Mass  {Garter's)  (Fearnley's  method). 


Carmine  . . . . .3  grams. 

Strong  ammonia  . . . . 6 cc. 

Glacial  acetic  aciil  . . . . 6 ,, 

Coignet’s  French  gelatine  . . .7  grms. 

Water  . , . . . 80  cc. 


Cut  up  the  gelatine  into  small  pieces  and  place  it  in  50  cc.  of 
the  water  to  swell  up,  z.e.,  for  four  or  five  hours.  Rub  up  the 
carmine  in  a mortar  with  a little  water  and  add  the  ammonia. 
Let  it  stand  for  two  hours  and  then  pour  it  into  a bottle,  rinsing 
the  mortar  with  the  remainder  of  the  water.  Place  the  swollen- 
up  gelatine,  and  any  remaining  water  unabsorbed  by  it,  on  a 
water-bath  until  it  melts.  To  the  dark  purple  carmine  fluid  add 
the  acid  (a  few  drops  at  a time),  mixing  the  two  thoroughly,  and 
as  soon  as  the  fluid  changes  to  a crimson  stop  adding  the  acid.  To 
the  melted  gelatine  add  the  crimson  carmine  little  by  little  and 
keep  stirring  all  the  time. 

This  mass  may  be  kept  in  a cool  place  for  a long  time  if  its 
surface  be  covered  with  methylated  spirit.  Before  using  it, 
dissolve  it  on  a water-bath,  and  filter  it  through  fine  flannel 
wrung  out  of  hot  water.  The  best  gelatine  to  use  is  French 
gelatine — Goignet’s. 

4.  Blue  Mass. — The  mass  is  made  with  gelatine  coloured  with 
soluble  Prussian  blue  or  Briicke’s  blue.  It  is  very  difficult  to 
obtain  a pure  sample  of  Briicke’s  blue,  but  this  can  now  be  had 
from  Dr.  Griibler  of  Leipzig.  Use  a saturated  watery  solution  of 
Briicke’s  blue. 

Weigh  5 grms.  of  gelatine,  and  treat  it  exactly  as  described 


INJECTING  BLOOD-VESSELS  AND  GLAND-TUBES. 


91 


for  the  carmine  mass  of  Ranvier.  Take  125  cc.  of  the  blue  solution 
and  heat  it  on  a water-bath,  and  when  the  gelatine  is  fluid  and 
still  on  the  water-bath,  add  the  warm  blue  solution  in  a small 
quantity  at  a time  and  stir  briskly.  The  glass  rod  should  show  no 
granules  on  it  when  it  is  withdrawn  from  the  mass.  Filter  the 
mass  through  new  flannel.  Even  the  best  gelatine  gives  a precipitate 
at  first,  but  it  disappears  with  heat. 

Other  injection  masses  are  used,  e.g.^  a watery  solution  of 
Eriicke’s  blue,  or  gelatine  and  silver  nitrate.  These  are  referred 
to  in  the  text. 

Brass  Syringe. — Many  good  injections  have  been  made  with  a 
brass  syringe.  , The  syringe  should  have  a long  barrel,  and  be 
warmed  by  repeatedly  sucking  up  hot  water  before  the  injecting 
fluid  is  drawn  into  it.  When  the  injection  mass  is  forced  into 
the  blood-vessel,  the  pressure  should  be  applied  steadily,  and  should 
not  be  so  great  as  to  rupture  the  small  blood-vessels. 

When  the  blood-vessels  of  an  animal  are  to  be  injected,  deeply 
narcotise  an  animal  with  chloroform,  e,g.^  a rabbit  or  a rat ; make 
a vertical  incision  through  the  skin  from  the  lower  part  of  the 
neck  to  the  ensiform  cartilage,  cut  through  the  sternal  cartilages, 
turn  up  the  breast-bone,  and  pull  the  sides  of  the  thorax  apart 
to  reveal  the  contents  of  the  cliest.  Open  the  pericardium  and 
make  a snip  into  the  right  ventricle.  Tie  a ligature  round  the 
upper  part  of  the  sternum  to  prevent  escape  of  the  injection 
through  divided  vessels.  Wash  the  blood  out  of  the  chest.  Snip 
off  the  apex  of  the  heart,  whereby  the  cavity  of  the  left  ventricle 
is  opened  into. 

Insert  a cannula  into  the  left  ventricle  and  push  its  nozzle  into 
the  aorta.  Tie  it  firmly  into  the  aorta  with  a stout  thread. 
Place  the  animal  in  a bath  in  warm  water  at  40°  C.  If  a syringe 
is  to  be  used,  by  means  of  a pipette  fill  the  cannula  with  the 
injecting  mass,  and  attach  the  syringe  and  force  the  mass  onwards 
into  the  blood-vessels.  This  is  done  by  slow,  steady  pressure. 
It  takes  fifteen  or  twenty  minutes  to  make  a good  injection. 
Any  sudden  increase  of  pressure  is  a})t  to  cause  rupture  of  blood- 
vessels and  consequent  extravasation  of  the  injection  mass.  We  can 
judge  when  a part  is  well  injected  by  the  colour  of  semi-trans- 
parent parts,  such  as  the  gums  or  the  skin.  They  must  be  deeply 
coloured  by  the  injection  mass  if  the  injection  is  successful. 

Continuous  Air  Pressure. — Most  frequently  injections  are  now 
made  by  continuous  air  pressure.  The  apparatus  used  should  consist 
of  a tin  trough  sufficiently  large  to  contain  the  animal  to  be  injected, 
and  contain  sufficient  water  to  cover  it.  The  water  is  kept  at 
40°  C.  by  means  of  a gas-burner  or  spirit-lamp.  In  the  same  trough 
are, placed  the  injection  masses  in  Wolff’s  bottles.  Each  Wolff’s 


92 


PRACTICAL  HISTOLOGY. 


bottle  is  connected  to  a large  air-chamber  into  which  water  can  flow 
from  the  water-tap,  and  thus  compress  the  air.  The  pressure  within 
this  cylinder  can  be  registered  by  means  of  a manometer.  The  com- 
pressed air  acts  on  the  surface  of  the  injection  mass  in  the  Wolff’s 
l3ottle,  and  forces  it  through  a tube  which  is  attached  to  the  cannula 
fixed  in  the  aorta.  The  large  cylinder  for  the  compressed  air  may  be 
made  of  tin,  or  one  of  the  large  stone  jars  used  by  spirit  merchants, 
or  a carboy  may  be  used. 

After  the  tissues  are  injected,  they  should  be  cooled  rapidly  by 
being  placed  in  running  water.  After  the  mass  is  completely  set 
the  injected  organs  arc  cut  into  small  pieces  and  hardened  in 
alcohol. 

The  methods  of  interstitial  injection  of  fluids  and  the  puncture 
methods  are  referred  to  in  the  text. 


XVI.— EXAMINATION  OP  FRESH  TISSUES  AND 
FLUIDS. 

In  examining  a fresh  tissue  or  organ  snip  off  a small  part  with 
scissors  and  tease  it  in  normal  saline,  or  one  of  the  indifferent  fluids 
mentioned  in  Chapter  III.  A convenient  plan  with  some  organs, 
6.^7.,  lymphatic  gland  or  liver,  is  to  make  a fresh  cut  and  scrape 
the  surface  with  the  blade  of  knife,  and  then  examine  the  scrapings. 

If  it  be  desired  to  study  the  tissue  elements,  it  may  be  placed  in 
one  of  the  macerating  media  mentioned  in  Chapter  IV.,  the  parti- 
cular fluid  selected  depending,  of  course,  on  what  object  is  sought 
to  be  obtained. 

If  it  be  desired  to  render  certain  parts  of  the  tissue  more  trans- 
parent, it  may  be  examined  in  glycerine. 

Again  acetic  acid  (1-2  per  cent.)  may  be  added  to  a fresh  tissue. 
It  has  the  double  action  of  making  connective  tissue  swell  up 
and  become  transparent,  thus  making  nuclei  more  evident,  while  it 
also  slirivels  the  latter  somewhat.  Albuminous  granules  are  dis- 
solved by  it,  while  oil  globules  are  not,  so  that  it  may  be  useful 
occasionally  in  determining  the  nature  of  the  granules  in  proto- 
plasm. Elastic  fibres  are  not  affected  by  it,  and  thus  can  readily 
be  distinguished  from  the  white  fibres  of  connective  tissue.  Groups 
of  micro-cocci  are  also  not  affected  by  it. 

The  tissue  may  be  stained  by  acetic  fuchsin.  This  is  made  as 
follows : — To  a 2 per  cent,  solution  of  acetic  acid  add  sufficient 
fuchsin  to  give  a saturated  red  colour  {Kahlden).  This  reagent  not 
only  makes  the  nuclei  visible,  but  it  stains  them  as  well. 

Sometimes  a weak  watery  solution  of  iodine  makes  the  outlines  of 


EXAMINATION  OF  FRESH  TISSUES  AND  FLUIDS.  93 


tissue  elements  more  distinct.  It  is  used  in  the  form  known  as 
Lugohs  Solution  diluted  with  water. 

Iodine  ......  i part. 

Potassic  iodide  . . . . . 2 ,, 

Water  ......  100  ,, 

Weak- Alkalies  (1-3  per  cent.)  dissolve  most  tissues  with  the 
exception  of  elastic  fibres,  pigment,  fat,  and  bacteria. 

The  vapour  of  osmic  acid  or  the  fluid  itself  (i  per  cent.)  may  he 
used.  It  blackens  fatty  particles. 

Finally,  the  tissue  may  be  stained  by  means  of  a watery  solution 
of  methyl-green,  methyl-violet  5B,  acetic  fuchsin,  or  methylene-blue 
in  the  form  of  Loffler’s  Methylene-blue. 

Concentrated  alcoholic  solution  of  methylene  blue  . 30  cc. 

Caustic  potash  (o.  10  p.c.).  ....  100  ,, 


Scheme  for  Living  or  Fresh  Objects. 

Harden  the  pieces  (§  IV.). 


Stain  in  bulk. 


Embed  (§  IX.). 
Make  sections. 


Fix  oh  slide  (§  XL ). 


Stain  the  sections  (§  XIL). 
Clarify  sections  (§  XIII.), 


Mount  sections. 


94 


PRACTICAL  HISTOLOGY. 


Fresh  Fluids  with  Suspended  Particles.— In  the  examination  of 
a fluid  for  suspended  particles,  bacteria,  such  as  cells,  membranes, 
<fcc.,  especially  if  these  be  few  in  number,  it  is  well  to  place  it  in  a 


X. 


Fig.  51.— Hand  Centrifuge  made  by  Muencke,  Luisen-strasse,  58,  Berlin,  N.W. 
It  costs  £3,  10s. 


conical  glass  and  allow  the  deposit  to  subside.  It  can  then  be 
removed  with  a glass  pipette. 

Centrifugal  Apparatus.— To  collect  the  sediment  or  suspended 
particles,  a centrifugal  apparatus  is  most  useful.  By  means  of  it 
the  deposit  can  readily  be  collected  at  the  bottom  of  a test-tube. 


EXAMINATION  OF  FRESH  TISSUES  AND  FLUIDS.  95 


Fig.  5 1 shows  a form  of  hand-centrifuge  devised  by  Litten  ^ and 
Muencke  of  Berlin.  The  ligure  is  reduced  to  ^ the  natural  size. 
One  revolution  of  the  wheel  B — its  teeth  fit  into  the  thread  of  the 
vertical  axis  S — causes  50  revolutions  of  the  disc  M with  the  4 glass 
tubes  G.  The  wheel  can  readily  be  turned  100  times  per  minute, 
which  gives  5000  revolutions  per  minute  for  the  disc.  Fig.  II.  shows 
the  disc  in  full  rotation,  and  fig.  III.  the  form  of  glass  vessel  used. 

By  means  of  this  instrument,  corpuscles  of  light  specific  gravity, 
such  as  blood-corpuscles,  albumen,  micro-cocci,  as  well  as  crystals, 
e.g,  oxalates,  can  readily  be  obtained  in  the  form  of  a sediment,  and 
it  is  therefore  especially  useful  for  the  investigation  of  the  deposits  in 
urine  and  exudations.  It  is  also  very  useful  for  obtaining  tubercle 
bacilli  from  sputum. 


S3moptical  Statement. 


Processes  required  for  Preparing  a Specimen  for  Microscopic  Ex- 
amination^ e.g.^  the  Spinal  Cord  of  a Dog  or  Cat  (Garbini). 

Cut  the  cord  into  pieces  about  2 centimetres  in  length,  wash  them 
in  normal  saline  to  remove  all  blood.  Place  them  in 


1.  Bichromate  of  potash  (2  p.c.)  . . . 10  days 

2.  Wash  in  running  water  . . . .12  hours 

3.  Alcohol  (50  p.c.)  .....  I day 

4.  „ (70  p.c.) 4 days 

5.  ‘ „ (90  p.c.) I day 

6.  ,,  (absolute)  .....  i day 

7.  In  xylol  or  chloroform  ....  12  hours 

8.  In  paraffin  on  warm  bath  ....  6-8  hours 

9.  Embed  in  paraffin. 

10.  Cut  sections  with  microtome,  and  fix  them  on  a slide' 

with  fixative.  1 

11.  Kemove  paraffin  by  washing  in  toluol  or  turpentine,  and  | 

then  in  absolute  alcohol  to  remove  essential  oil. 


bo 


w 


. 

a ^ 

bb 

a 


a 

O 


12.  Place  in  70  p.c.  alcohol. 

13.  Stain  in  strong  carmine 

or  haematoxylin  (5-15 
minutes). 

14.  Wash  in  water,  then  in 

70  p.c.  alcohol,  and  de- 
hydrate in  absolute  alco- 
hol (5-10  minutes). 


§ ^ 
ra  *3? 

^ S 
^0  g) 
a <D 
*a  > 


CO 


12.  Place  in  distilled  water. 

13.  Pour  on  a few  drops  of 

the  aniline  dye  (3-5 
minutes.) 

14.  Wash  in  water  and  then 

in  absolute  alcohol  until 
the  section  has  the  de- 
sired tint. 


^ Deutsch,  med.  Wochensch.y  No.  23,  1891. 


o6 


PRACTICAL  HISTOLOGY. 


15.  After  removing  surplus  alcohol,  pour  on  a few  1 Dehydra- 

drops  of  essential  oil  (origanum,  xylol).  > tion  and 

16.  Remove  oil  and  mount  in  xylol-balsam.  ) mounting. 

N.B. — In  making  a balsam  preparation,  the  sections  must  always 
pass  through  four  groups  of  fluids. 

I > 2 > 3 ^ > 4 

Watery  liquid — Alcohol — Essential  oil — Balsam. 

I < 2 < 3 < 4 

Never  take  an  object  from  one  group  to  another  of  the  series 
without  passing  it  through  the  intermediate  group;  it  must  be  passed 
from  the  ist  to  the  4th,  and  on  the  return  from  the  4th  to 
the  ist. 


PART  IL 


LESSON  I. 

MILK,  GRANULES,  FIBRES,  AND  VEGETABLE 
ORGANISMS. 

1.  Examine  the  Microscope,  the  objectives,  and  the  eye-pieces. 

(a.)  Select  the  objective  and  ocular  required.  For  a high  power 
(H),  if  a Zeiss’  microscope  be  used,  select  the  objective  D and  the 
ocular  2 ; if  Hartnack’s,  the  objective  No.  7 and  the  eye-piece  III. 
See  that  the  lenses  are  clean.  Place  the  ocular  in  the  tube,  and 
screw  the  H lens  to  the  lower  end  of  the  tube,  and  leave  it  half  an 
inch  above  the  level  of  the  stage.  For  a low  power  (L)  use  No.  2 
ocular  of  Zeiss  or  III.  of  Hartnack,  and  objective  A or  No.  3 
respectively.  In  using  a low  power,  the  lens  must  be  i|-  inches 
above  the  stage  to  begin  with. 

(^/.)  With  the  microscope  in  front  of  you,  with  high-power  lens 
on  it,  arrange  the  concave  side  of  the  mirror  under  the  stage  so  as 
to  reflect  a beam  of  light  up  the  tube  of  the  microscope  into  the  eye, 
looking  in  at  the  ocular.  Turn  the  sub-stage  diaphragm  until  a 
small  aperture  in  it  is  under  the  aperture  in  the  centre  of  the  stage. 
If  any  specks  are  visible  on  looking  througli  the  microscope,  rotate 
the  ocular  ; if  they  move,  of  course  they  are  on  the  ocular  itself. 
Clean  the  outer  surfaces  of  the  lenses  of  the  ocular  with  a piece  of 
clean  wash-leather,  which  should  be  kept  tied  to  the  microscope  and 
used  for  no  other  purpose  than  cleaning  the  lenses.  Replace  the 
ocular,  and  if  specks  are  still  present  and  move  when  the  ocular  is 
moved,  they  must  be  on  the  inner  surface  of  the  eye-glass  or  field- 
glass  of  the  latter.  This  is  easily  determined  by  rotating  the  eye- 
glass of  the  ocular  alone,  while  looking  through  the  microscope,  and 
observing  if  the  specks  do  or  do  not  move  with  it.  Clean  the  inner 
surfaces  of  these  lenses.  A general  dimness  indicates  that  the  objec- 
tive itself  is  dirty.  The  light  used  should  not  be  direct  sunlight, 
but  preferably  light  reflected  from  a white  cloud. 

10 


G 


98 


PRACTICAL  HISTOLOGY. 


L- 

The  rule  with  regard  to  the  use  of  the  diaphragm  must  never  be 
neglected,  viz.,  to  use  a small  aperture  with  a high  power,  and  a 
large  aperture  with  a low  power. 

2.  Clean  a Slide  and  Cover-Glass. 

(a.)  The  Slide. — Seize  the  slide  by  its  edges  with  the  thumb  and 
forefinger  of  the  left  hand,  dip  one  half  of  it  into  water,  withdraw 
it,  and  with  a clean  old  handkerchief  rub  both  wetted  surfaces  at 
once  until  they  are  clean  and  dry.  Reverse  the  slide,  still  holding 
it  by  its  edges,  and  dip  the  other  end  in  water,  and  clean  its  surfaces 
as  before.  Lay  the  slide  upon  some  clean,  suitable  background, 
white  or  black  paper,  or  on  the  photophore. 

{h.)  The  Cover-Glass. — Sometimes  the  covers  have  a thin  film  on 
them ; this  may  be  got  rid  of  by  placing  them  in  strong  sulphuric 
acid,  and  subsequently  removing  every  trace  of  acid  by  water.  Dip 
the  cover-glass  in  water,  take  it  between  two  folds  of  a handkerchief 
held  between  the  thumb  and  forefinger  of  the  right  hand,  and  rub 
both  surfaces  at  once.  After  it  is  cleaned,  do  not  lay  it  flat,  but 
tilt  it  up  against  some  convenient  object. 

The  first  lesson  is  devoted  to  the  examination  of  a few  simple 
objects — some  of  which  are  occasionally  found  as  foreign  bodies  in 
microscopical  preparations — with  a view  to  familiarise  the  student 
with  the  use  of  the  microscope. 

3.  Milk.— By  means  of  a glass  rod,  place  on  the  centre  of  the 
slide  a small  drop  of  milk  diluted  with  three  or  four  volumes  of 
water.  To  find  the  centre  of  the  slide,  use  the  mounting  block 
(p.  89). 

Apply  a Cover-Glass. — Seize  the  cover-glass  by  the  edge  by  means 
of  a pair  of  forceps  with  broad  points.  The  pattern  shown  in  fig. 
6 is  convenient.  The  edge  of  the  cover-glass  opposite  to  the  forceps 
is  allowed  to  touch  the  slide  close  to  the  drop  of  fluid,  the  edge 
opposite  being  gradually  and  evenly  lowered  by  depressing  the 
forceps  until  the  fluid  touches  the  under  surface  of  the  cover-glass. 
By  lowering  the  cover-glass  thus  gently  and  obliquely  the  entrance 
of  air-bubbles  is  avoided.  Place  the  object  on  the  stage  right  under 
the  lens. 

Focus  the  Object  (H). — The  objective  is  still  half  an  inch  above 
the  stage.  While  looking  into  the  eye-piece  of  the  microscope, 
seize  the  tube  of  the  latter  between  the  thumb  and  adjoining  fingers 
of  the  right  hand,  and  with  a screwing  or  twisting  movement  of 
the  tube  from  left  to  right,  gradually  depress  the  tube  until  the 
outlines  of  the  object  are  indistinctly  seen.  This  is  the  coarse 
adjustment.  The  focussing  process  is  facilitated  by  keeping  the 
slide  and  object  moving  slightly.  This  can  readily  be  done  l)y 
moving  the  slide  with  the  thumb  and  forefinger  of  the  left  hand, 
the  ulnar  margin  of  the  palm  conveniently  resting  on  the  table. 


l]  MILK,  FIBRES,  VEGETABLE  ORGANISMS.  99 

Xow  use  iYie  fine  adjustment,  and  bring  the  outlines  of  the  object 
in  the  field  sharply  into  view. 

It  is  of  the  greatest  importance  that  the  student  should  be  taught 
to  describe  the  objects  which  he  sees,  and  also  to  make  sketches  of 
them.  To  facilitate  the  description  of  isolated  objects,  the  follow- 
ing heads  may  be  ado|3ted  : — 

a.  Shape. 

h.  Border. 

c.  Surfaces  (upper  and  lower). 

d.  Size. 

e.  Colour. 

/.  Transparency  and  relation  to  light. 

g.  Contents. 

h.  Effects  of  reagents. 

In  the  object  under  examination  there  is  a large  number  of 
minute  bodies  floating  in  a fluid.  Describe  the  appearance  of  the 
floating  particles  under  the  following  heads  : — 

(a.)  Shape. — The  milk  globules  (fig.  52)  are  spherical,  as  can  be 
shown  by  touching  the  edge  of  the  cover-glass  with  a needle,  and 
then  observing  them  as  they 
rotate  in  the  field  of  the  micro- 
scope. Moreover,  if  one  be 
focussed,  its  outline  comes 
gradually  into  focus,  and  dis- 
appears gradually,  while  optically 
with  regard  to  light  these  bodies 
behave  as  globules,  and  not  as 
discs. 

(6.)  Border.  — Smooth  and 
regular. 

(c.)  Surfaces. — Elevate  the 
objective  by  means  of  the  fine 
adjustment  until  the  upper  sur- 
face of  the  globules  comes  into 
view ; depress  it  again  slowly 
and  then  examine  the  globule 
throughout  its  entire  thickness,  until  its  under  surface  is  brought 
into  view.  Both  surfaces  are  smooth. 

{d.)  Size. — The  globules  are  not  all  of  the  same  size.  If  desired, 
measure  their  actual  size  (p.  20). 

{e.)  Colour. — The  smaller  ones  appear  colourless,  but  some  of  the 
larger  may  have  the  slightest  tinge  of  a faint  yellow. 

If.)  Transparency  and  relation  to  light — They  are  transparent, 
because  the  outline  of  a subjacent  one  can  be  seen  through  a globule 


PRACTICAL  HISTOLOGY. 


lOO 


[I- 


lying  over  it.  ^N’otice  also  the  highly  retractile  character  of  each 
globule,  characteristic  of  an  oil  droplet  (fig.  55,  3). 

{g,)  Contents, — They  appear  homogeneous  and  uniform,  and  no 
included  body  is  to  be  seen.  Each  globule  is,  in  fact,  a globule 
of  oil. 

(/?.)  Effects  of  reagents. — To  one  side  of  the  cover-glass  apply  a 
drop  of  acetic  acid.  To  the  opposite  edge  of  the  cover-glass  apply 
the  apex  of  a triangular  piece  of  blotting-paper.  The  blotting- 
paper  sucks  up  some  of  the  milk,  and  the  acid  runs  in  at  the 
opposite  side  to  supply  its  place.  This  is  the  process  of  irrigation. 
Move  the  slide  to  bring  into  focus  a part  of  the  field  which  has 
been  acted  on  by  the  acid,  and  note  that  the  corpuscles,  instead  of 
floating  about  singly  as  before,  are  now  aggregated  into  small  groups. 
The  acid  seems  to  have  altered  the  surfaces  of  the  globules,  so  that 
they  adhere  to  each  other.  The  acid  is  said  to  act  on  the  casein 
envelopes  of  the  globules,  and  to  soften  or  dissolve  them.  This 
preparation  is  not  to  be  preserved. 

Make  sketches  of  these  objects  before  and  after  the  action  of 
reagents. 

4.  Potato-Starch  Granules. — With  the  blade  of  a knife  gently 
scrape  the  surface  of  a freshly-cut  raw  potato ; place  the  matter 
so  obtained  in  a drop  of  water  on  a slide.  Kemove  any  coarse 
fragments,  and  apply  a cover-glass.  Focus  the  object  (H). 

{a.)  Observe  that  the  granules  (fig.  53)  are  ovoid  bodies  of 
unequal  size,  not  equal  at  the  two  ends,  clear,  and  with  a sharp 
outline,  ^^ear  the  smaller  end  of  each  granule 
notice  a small  spot,  the  “ nucleus  ’’  or  hilum,  round 
which  are  concentric  layers,  giving  rise  to  the 
appearance  of  fine  concentric  lines  arranged  with 
relation  to  the  nucleus.  The  lines  are  more 
numerous  on  one  side  of  the  hilum  than  the  other. 
Sketch  two  or  three  of  the  granules. 

(/>.)  Irrigate  the  corpuscles  with  a diluted  solu- 
tion of  iodine  in  iodide  of  potassium.  Each 
granule  becomes  blue  as  the  iodine  reaches  it. 
This  IS  due  to  the  formation  of  iodide  of  starch. 
If  the  iodine  be  too  strong  the  granules  appear  black. 

5.  Rice-Starch  (H). — Examine  a little  rice-starch  in  the  same 
way.  [NTotice  the  much  smaller  irregular  granules.  Each  granule 
is  polygonal,  mostly  five  or  six  sided.  The  granules  are  stained  blue 
by  iodine  (fig.  54).  ^lake  sketches  of  the  starch  corpuscles. 

6.  Gamboge  and  Brownian  Movement  (H). — Rub  up  a small 
piece  of  solid  gamboge  in  water  until  the  latter  has  a faint  yellow 
appearance.  Place  a drop  on  a slide,  cover,  and  examine. 

(a.)  Observe  granules  of  various  sizes  and  shapes  floating  in  the 


I.]  MILK,  FIBRES,  VEGETABLE  ORGANISMS.  lOI 

field.  The  larger  ones  appear  coloured  yellow,  but  the  finer  ones 
cannot  be  seen  distinctly  as  yellow  bodies. 

(h.)  Note  the  finer  granules,  which  exhibit  a slow 
dancing  movement.  They  are  never  at  rest.  This  is 
called  Brownian  movement,  and  appears  to  be  due  to 
inequalities  of  temperature  in  different  strata  of  the 
fluid.  This  movement  is  exhibited  by  all  finely- 
divided  particles  suspended  in  a fluid,  e.y.,  China  ink, 

Berlin  blue,  provided  the  particles  be  small  enough, 
and  the  fluid  in  which  they  are  suspended  be  not  too 
viscid.  The  fine  granules  in  salivary  corpuscles  ex- 
hibit this  movement.  (Lesson  IV.  1,  d). 

7.  Air-Bubbles  in  Gum  or  Water  (H). — Make  a solution  of  gum 


Fig.  54. — Rice- 
Starch. 


Fig.  55. — I.  Bubble  of  air  in  water  (A)  when  the  lower  surface  is  in  focus  ; (B)  Middle,  and 
(C)  Upper  surface  of  focus.  2.  Bubble  of  air  in  Canada  balsam.  3.  Globule  of  oil  in 
water, 


102 


PRACTICAL  HISTOLOGY. 


[I. 


mucilage  and  shake  it  up  in  a test-tube  with  air  until  it  forms  fine 
bubbles.  Place  a drop  of  it  with  its  included  bubbles  on  a slide, 
cover,  and  examine. 

(«.)  Observing  larger  and  smaller  bubbles,  especially  note  how 
the  appearance  of  the  bubble  varies  with  the  elevation  or  depres- 
sion of  the  lens.  Sketch  these  appearances.  When  the  tube  is 
depressed,  the  bubble  has  a small,  clear  centre,  and  a wide,  black, 
sharp,  retractile  margin  (fig.  55,  A),  because  so  much  of  the  light 
is  refracted  by  the  air,  and  does  not  pass  through  the  bubbles  of 
air  into  the  lens.  Study  the  appearance  of  the  bubble  when  the 
centre  and  then  the  upper  surface  are  in  focus  (B,  C). 

The  student  will  have  an  opportunity  by-and-by  of  observing 
the  appearance  of  a bubble  of  air  in  Canada  balsam  (fig.  55,  2),  and 
an  oil  globule  in  water  (fig.  55,  3). 

8.  Cotton  Fibres  (H). — Place  a few  fibres  of  cotton-wool  in 
water,  cover,  and  examine. 

(a.)  Observe  the  fine  translucent  flattened  threads,  which  are 
really  tubes,  each  looking  as  if  twisted  on  itself  at  intervals 

(fig-  56)- 

(h.)  Eemove  the  cover-glass  and 
the  water,  add  a drop  of  iodine ; 
cover  and  examine  j note  that  the 
fibres  are  yellowish ; add  a drop  of 
strong  sulphuric  acid  or  a drop  of  the 
following  mixture  : — Glycerine  2, 
water  i,  and  sulphuric  acid  3 parts. 
At  the  edge  of  the  cover-glass  suck 
it  through  with  blotting-paper,  and 
note  that  the  fibres  become  blue. 
They  are  composed  of  cellulose, 
which  does  not  give  a blue  with 
iodine  alone,  but  with  iodine  and 
sulphuric  acid. 

9.  Linen  Fibres  (H). — Examine 
in  water. 

(a.)  Observe  the  cylindrical  or 
flattened  translucent  fibres  with  no 
twist ; but  they  have  a few  markings  on  them  here  and  there,  and 
at  these  points  the  fibre  is  generally  slightly  thicker.  They  are  in 
reality  tubes  with  thick  walls  (fig.  56). 

10.  Wool  (H).  — Examine  in  water. 

(a.)  Observe  the  cylindrical  fibres  with  numerous  zigzag  trans- 
verse lines  due  to  the  epithelial  covering  of  the  fibre  (fig.  56). 
It  is  convenient  to  examine  dyed  wools  also,  as  some  of  these 
exhibit  the  zigzag  imbricate  scales  even  better  than  undyed  wool. 


Silh,  Wool.  Linen.  Cettent 

Fig.  56.— Fibres  of  Silk,  Wool,  Cotton, 
and  Linen. 


I-] 


MILK,  FIBRES,  VEGETABLE  ORGANISMS. 


103 


11.  Vegetable  Cells  forming  a Membrane. — With  a pair  of 
forceps  peel  off  a thin  layer  of  the  covering  of  a fresh  onion  bulb. 
* Examine  it  either  in  water  or  in  water  after  staining  with  a solution 
of  iodine  (H).  Observe  the  cells  (fig.  57)  with  well-defined  walls, 


Nn  N V 


Fig.  57. — Cells  from  bulb  of  a fresh  onion  forming  a membrane.  M,  cell  membrane ; 

N,  nucleus  seen  from  the  surface;  Nn,  nucleolus;  V,  vacuoles,  x 240. 

and  united  to  each  other  by  the  edges  to  form  a membrane — the 
excentrically  placed  spherical  nucleus  and  the  granular  cell-con- 
tents. 

12.  Cells  with  sinuous  margins,  and  containing  chlorophyll 
granules,  may  be  studied  in  a leaf  of  the  Duckweed  or  Lemna  minor ^ 
also  the  reaction  for  cellulose  (sulphuric  acid  and  then  tincture  of 
iodine  = blue)  in  the  same  plant 

13.  The  currents  in  protoplasm  are  well  seen  (H)  in  Cliara  vul- 
garis^ which  is  so  common  in  our  streams. 

Vegetable  Micro-Organisms. — These  are  frequently  found  in 
microscopic  preparations,  while  others  are  the  cause  of  various 
diseases.  They  are  usually  classified  as  follows : — 

1 . Moulds. 

2.  Saccliaromgcetes  (yeast-like  organisms). 

3.  Scliizomgcetes  (bacteria-like  organisms). 

14.  Penicillium  (H). — This  mould  is  readily  found  on  starch- 
paste  or  on  Muller’s  fluid  preparations  which  have  been  left  stand- 


PRACTICAL  HISTOLOGY. 


104 


[I. 


Fig.  58.— Deve- 
lopment of  a 
Gonidiophore 
bearing  Stylo- 
gonidia  (st.g) 
on  the  Hyphfe 
of  Penicil- 
lium. 


ing  uncovered.  Place  a little  on  a slide,  add  a drop  of  water,  cover, 
and  examine. 

(a.)  Observe  the  stems,  consisting  of  narrow,  clear,  oblong  cells, 
joined  end  to  end,  and  on  the  summit  of  each  are  several 
rows  of  small  spores.  The  rootlets  or  mycelium  consist 
of  elongated  chains  of  narrow  oblong  cells. 

The  mycelium  is  composed  of  much-branched  liyphce. 
The  cells,  elongated  and  narrow,  composing  these  are 
separated  from  each  other  by  numerous  partition  walls 
(fig.  60,  e).  The  walls  consist  of  cellulose  and  contain 
protoplasm  with  vacuoles. 

(h.)  Search  for  one  of  the  hyphse  which  bear  on 
their  free  ends  a brush-like  group  of  cells,  some  of 
which  become  constricted  to  form  chains  of  spherical 
stylogonidia  (fig.  58).  The  ripe  stylogonidia  have  a 
green  colour,  and  they  give  the  mould  its  green 
appearance. 

15.  Yeast  (H). — Place  some  German  yeast  in  sugar 
and  water ; keep  it  for  several  hours  in  a warm  place.  Examine  a 
drop  of  the  fluid. 

(a.)  Observe  the  small  oval  yeast-cells,  each  with  an  envelope,  a 
^ ^ ^ large  clear  vacuole,  and  granular  proto- 

plasm. 

(hi)  Search  for  one  budding,  and 
notice  the  small  faintly  granular  bud 
adhering  to  and  projecting  from  the 
mother-cell  (figs.  59,  60,/). 

Each  yeast  plant  is  a single  cell  or 
It  is  composed  of  a transparent  thin  delicate 
composed  of  cellulose.  When  the  cell  is 

ruptured  the  empty  cell-envelope  may 
be  seen  in  the  field.  Within  is 

granular  'protoplasm,  containing,  as  a 
rule,  a clear  space  or  vacuole.  Some 
cells  contain  a nucleus. 

(c.)  Stain  the  preparation  with  a 
watery  solution  of  magenta.  Note 
that  all  the  cells  do  not  stain  equally 
well.  The  buds  on  the  side  are  stained 
of  a deep  red,  and  to  a less  extent  the 
protoplasm  of  most  of  the  cdls. 

16.  Micrococci  and  Bacteria  ( H ). — 
Set  aside  a solution  of  peptone  or  a watery  extract  of  a piece  of  flesh 
for  some  time,  until  a scum  forms  on  the  surface  due  to  putrefaction. 
Place  a little  of  the  scum  on  a slide,  cover,  and  examine. 


Fig.  59.— Yeast-Cells,  a.  h and  c 
Budding. 


morphological  unit, 
envelope  or  cell-wall, 


Fig.  6o.—d.  Micrococci  in  cylinders ; 
e.  Mycelium  of  Prnicillium ; /. 
Yeast-Cells;  g.  Bacilli  and  Micro- 
cocci. 


1.]  MILK,  FIBRES,  VEGETABLE  ORGANISMS.  IO5 

(a.)  Observe  groups  of  small  round  specks,  often  held  together 
by  a homogeneous  medium.  These  are  micrococci  (fig.  60,  d). 

(b.)  Small  elongated  rod-like  bodies,  each  moving  across  the  field 
in  a zigzag  like  manner.  These  are  bacteria  (fig.  60,  g). 

— In  all  cases  sketrhes  must  he  made  of  the  objects  examined. 
This  holds  good  for  this  and  all  succeeding  lessons, 

17.  Determine  the  magnifying  power  of  the  microscope. — Do 
this  according  to  the  method  described  at  p.  19. 


ADDITIONAL  EXERCISES. 

18.  Staining  of  Fission  Fungi  or  Schizomycetes.  — They  stain  readily  with 
aniline  dyes,  especially  basic  aniline  colours.  Scrape  off  a little  of  the  coating 
which  accumulates  on  the  surface  of  the  molar  teeth,  press  it  between  two  cover- 
glasses,  so  as  to  make  cover-glass  preparations.  Glide  the  one  glass  off  the 
other.  Place  the  cover-glasses,  film  surface  downwards,  in  a i per  cent,  solu- 
tion of  methyl-violet,  gentian -violet,  or  methylene-blue  contained  in  a watch- 
glass.  Heat  the  watch  glass  and  its  contents  over  a gas-flame  until  a faint 
cloud  of  vapour  rises  ; allow  it  to  cool,  and  in  5-10  minutes  or  so  the  colora- 
tion is  complete.  Remove  the  cover-glasses,  wash  them  in  water,  and  place 
them  in  absolute  alcohol.  Remove  the  cover-glasses  apd  allow  them  to  dry  in 
the  air  and  mount  in  xylol  balsam.  A moist  cover-glass  preparation  from 
absolute  alcohol  may  be  clarified  with  xylol. 

{a.)  Observe  many  epithelial  cells  coloured,  but  so  are  the  organisms. 

If  it  is  desired  to  ha'^'e  only  the  organisms  coloured,  the  stained  cover-glasses 
are  taken  direct  from  ^he  staining  solution,  or  laved  in  absolute  alcohol  and 
placed  in  a solution  of  iodine  composed  of  i part  iodine,  2 parts  ]>otassic  iodide, 
and  300  water,  in  which  they  are  kept  for  a few  minutes.  Transfer  them  to 
and  wash  them  in  absolute  alcohol  until  nearly  all  colour  is  gone,  and  clarify 
with  xylol.  Only  the  organisms  are  of  a dark-blue  tint  ; the  other  tis.«ues  are 
decolorised  or  nearly  so.  This  is  Gram’s  method. 

Stain  bacteria  from  a putrefying  proteid  fluid,  e,g.,  peptones  01  meat- 
extract,  in  the  same  way. 


11 


io6 


PRACTICAL  HISTOLOGY. 


[II. 


LESSON  II. 

THE  BLOOD. 

Under  the  microscope  blood  is  seen  to  consist  of  a clear,  transparent 
fluid,  the  plasma  or  liquor  sanguinis,  in  which  are  suspended  the 
blood-corpuscles.  The  blood-corpuscles  are  of  two  kinds,  the  red 
or  coloured,  and  the  white,  pale,  or  colourless.  Besides  these, 
shere  fall  to  be  examined  the  blood-plates,  or,  as  they  are  also 
called,  blood-tablets  or  platelets. 

f Blood-plasma. 

Blood  -j  I Red. 

t Corpuscles  I White. 

( Platelets. 

BLOOD-CORPUSCLES  OF  AMPHIBIANS. 

(A.)  Coloured  Corpuscles  of  Amphibians  (Newt  or  Frog). — After 
destroying  the  brain  of  a newt  or  frog,  a drop  of  blood  may  be 
obtained  from  the  cut  end  of  the  tail  of  the  former,  or  from  the  cut 
surface  after  amputation  of  the  foot  of  the  latter. 

1.  Red  Corpuscles  (H). — Place  a small  drop  of  blood  in  the 
centre  of  a perfectly  clean  slide  and  cover  it  at  once  with  a cover- 
glass.  Examine  it  with  a high  power.  To  avoid  the  pressure  of 
the  cover-glass,  a short  length  of  a hair  may  be  placed  in  the  blood 
droplet  before  the  cover-glass  is  applied. 

(a.)  Observe  the  coloured  and  colourless  corpuscles,  the  former 
much  more  numerous  than  the  latter  (fig.  6i). 

(h.)  Study  the  red.  corpuscles.  Observe  that  they  are  very  numer- 
ous, elliptical  in  outline  when  seen  on  the  flat,  slightly  yellowish 
in  colour  ; their  border  or  contour  is  even  and  well  defined.  Select 
one  seen  on  edge,  and  note  that  it  is  a thin  ellipse,  pointed  at  the 
ends,  becoming  gradually  thicker  in  the  centre,  so  that  it  is  a hi-convex 
elliptical  disc.  Sometimes  one  corpuscle  can  be  seen  overlying  a 
subjacent  one,  in  which  case  the  outline  of  the  latter  can  be  dis- 
tinctly seen  through  the  former,  indicating  that  the  corpuscles  are 
transparent  (fig.  6i).  Notice  within  each  corpuscle  a lighter  oval, 
central  area,  indicating  the  existence  of  an  elliptical,  colourless. 


TI.l 


THE  BLOOD. 


107 


elongated,  granular-looking  included  body — tire  nucleus.  The  long 
axis  of  the  colourless  nucleus  coincides  with  the  long  axis  of  the 
corpuscle.  At  first,  the  nucleus 
may  not  be  very  distinct,  but 
after  a time  it  becomes  distinctly 
visible,  and  it  can  be  readily 
made  so  by  the  action  of  certain 
reagents,  especially  weak  acids. 

Small  vacuoles  frequently  appear 
in  the  body  of  the  corpuscle,  more 
especially  in  frogs  that  have  been 
kept  some  time.  In  others  there 
may  be  seen  a faint  radiate 
striping  of  the  corpuscles  as  if 
there  were  fine  folds  in  it  due  to 
partial  drying. 

The  yellow  colour  is  due  to  the 
presence  of  haemoglobin,  which 
is  enclosed  within  the  meshes  of 
a colourless  stroma  or  delicate 
framework. 

If  the  blood  be  taken  from  a 
frog  which  has  been  kept  through- 
-out  the  winter,  vacuoles  may  be 
seen  in  the  haemoglobin  of  the  red  corpuscles  (fig.  61,  v).  They  are 
rapidly  produced  in  frogs  after  the  injection  of  ammonium  chloride. 

Sketch  two  or  three  corpuscles,  both  red  and  white  ; the  red  cor- 
puscles both  on  the  flat  and  on  edge,  and  two 
overlapping  each  other. 

2.  Acetic  Acid. — To  the  edge  of  the 
cover-glass  of  the  same  preparation  apply  a 
drop  of  dilute  acetic  acid  (i  per  cent. — i cc. 
glacial  acetic  acid  to  99  cc.  of  normal  saline 
solution).  The  acid  runs  in  under  the  cover- 
glass,  but  if  it  does  not,  apply  a small  tri- 
angular piece  of  blotting-paper  to  the  opposite 
edge  of  the  cover-glass.  Let  one  of  the  angles 
of  the  blotting-paper  touch  the  fluid  under 
the  cover-glass,  and  it  will  suck  up  some  of 
the  blood,  and  thus  cause  the  acetic  acid  to 
run  in  at  the  opposite  side.  This  is  known 
as  the  process  of  irrigation.  Move  the  slide,  and  find  a part  of  the 
object  which  has  been  acted  on  by  the  dilute  acid. 

(a.)  Observe  that  the  red  corpuscles,  or  at  least  most  of  them, 
become  spherical  and  decolorised,  the  nucleus  becomes  very  distinct 


Fig.  61.— Blood  of  Frog.  a.  Red  corpuscle 
seen  on  the  flat ; h.  In  Profile ; c.  Three- 
quarter  face  ; some  of  the  red  corpuscles 
show  the  presence  of  vacuoles  (v) ; n. 
Colourless  corpuscle  at  rest;  rw.  One 
with  amoeboid  processes ; p.  Fusiform 
cell,  probably  from  the  vascular  wall. 


Fig.  62. — Amphibian  Coloured 
Blood -Corpuscles  seen  on 
the  Flat  and  on  Edge, 
X 1000. 


I08 


PRACTICAL  HISTOLOGY. 


[II. 


and  granular,  while  the  outline  of  the  corpuscle  may  become  very 
indistinct  (fig.  63).  The  nucleus  appears  somewhat  shrunken  or 
shrivelled.  If  the  corpuscles  are  roughly  treated,  the  nucleus  may 
be  seen  placed  excentrically,  or  even  extruded  from  the  corpuscle. 
The  acid  acts  on  the  haemoglobin,  forming  a nevv  compound^  which 
diffuses  out  of  the  corpuscles  and  stains  the  surrounding  plasma  a 
faint  yellow.  The  nucleus  in  some  of  the  corpuscles  may  absorb 
some  of  the  yellow  pigment,  and  become  stained  thereby,  especially 
if  a strong  solution  of  acid  has  been  used.  The  action  on  the 
colourless  corpuscles  is  referred  to  at  p.  112. 

3.  Dilute  Hydrochloric  Acid,  i per  cent.  (H). 

(a.)  To  a fresh  drop  of  blood  add,  as  before,  a drop  or  two  of 
dilute  hydrochloric  acid.  Watch  diligently  one  or  two  of  the  red 
corpuscles.  They  gradually  enlarge,  become  spherical,  and  may  all 
of  a sudden  burst  and  discharge  their  contents,  the  nucleus  coming 
clearly  into  view  during  the  process.  After  the  rupture,  the  residue 
of  the  stroma  of  the  corpuscles  may  be  seen  in  the  field.  In  other 
cases  the  corpuscles  become  clear,  globular,  and  transparent,  with 


Fig.  63.— Frog’s  Red 
Blood  - Corpuscle 
acted  on  by  Dilute 
A.cetic  Acid,  x 300. 


Fig.  64.  — Action  of 
Water  on  an  Am- 
phibian Coloured 
Blood-Corpuscle. 


Fig.  65. — Action  of 
Syrup  on  Frog’s 
Red  Blood  - Cor- 
puscle, X 300. 


here  and  there  fine  shreds  stretching  between  the  nucleus  and  the 
surface  of  the  spherical  corpuscle. 

4.  Water  (H). — To  a fresh  preparation  of  blood  apply  a drop  of 
water  to  the  edge  of  the  cover-glass,  and  notice  its  effects  upon  the 
corpuscles. 

(a.)  The  water  rapidly  diffuses  into  the  corpuscles  and  renders 
them  spherical,  while  at  the  same  time  it  decolorises  them,  the 
haemoglobin  diffusing  outwards  into  the  plasma,  and  staining  it 
slightly  yellow.  The  nucleus  also  becomes  spherical.  Thus  the 
outline  of  the  corpuscles  be  ‘omes  very  faint  in  the  field  of  the 
microscope,  the  corpuscles  themselves  now  almost  consisting  of  a 
nucleated  stroma  (fig.  64). 

5.  Strong  Syrup  (H). — Place  a small  drop  of  blood  on  a slide 
and  near  it  a drop  of  syrup ; mix  the  two  with  a needle,  and  apply 
a cover-glass. 

(a.)  Observe  that  some  of  the  red  corpuscles  are  rapidly 
shrivelled  and  puckered,  especially  when  seen  on  edge,  owing  to 
fluid  passing  out  of  them  by  exosmosis  (fig.  65).  Some  of  them 


II.] 


THE  BLOOD. 


109 


may  present  here  and  there  a reddish  tinge.  All  the  corpuscles 
are  not  affected  equally  or  at  the  same  time.  The  same  effects  arc^ 
produced  by  strong  saline  solutions. 

6.  Tannic  Acid  (H). — On  a slide  mix  a drop  of  blood  with  a 
drop  of  tannic  acid,  using  a relatively  large  drop  of  the  acid  fluid ; 
wait  for  about  a minute,  and  then  apply  a cover-glass. 

{a.)  Observe  that  some  of  the  corpuscles  become  globular,  while 
the  haemoglobin  passes  out  of  the  corpuscles  at  one  or  more  spots, 
and  appears  on  the  surface  in  the  form  of  one  or  more  small 
granular  buds  (fig.  66,  c).  Sometimes  the  bud  or  buds  are  small. 
At  others  the  bud  may  be  as  large  as  the  remainder  of  the  corpuscle, 
which  has  become  smaller  and  partly  decolorised.  In  other  cases 
it  may  be  collected  around  the  nucleus  {Robert'^).  The  tannic  acid 
causes  a separation  of  the  haemoglobin  from  the  stroma. 

The  solution  of  tannic  acid  is  made  by  dissolving  2 grains  of 
tannic  acid  in  i oz.  of  boiling  water,  and  allowing  it  to  cool. 


7.  Boracic  Acid. — Mix  a drop  of  newt’s  or  salamander’s  blood 
with  a 2 per  cent,  solution  of  boracic  acid,  which  takes  a short 
time  to  act  on  the  corpuscles  arid  produce  its  effects. 

(a.)  Observe  that  the  haemoglobin  is  collected  around  the  nucleus, 
so  that  the  presence  of  the  latter  is  obscured,  but  in  many  of  the 
corpuscles  fine  threads  of  haemoglobin  remain  attached  to  the 
circumference  of  the  corpuscle,  so  that  the  retracted  haemoglobin 
may  have  a stellate  form,  while  the  rest  of  the  corpuscle  is  colour- 
less. To  the  latter  Briicke  gave  the  name  Oilwid,  to  the  former 
Zooid  (fig.  67). 

If  these  corpuscles  are  acted  on  for  twenty-four  hours  with  i per 
cent,  osmic  acid,  they  are  ‘‘  fixed,”  and  may  be  mounted  permanently 
in  glycerine-jelly. 

8.  Magenta. — T^Iix  a drop  of  blood  and  a drop  of  the  special 
magenta  fluid  (p.  74)  on  a slide,  cover,  and  examine. 

(a.)  Observe  that  the  nuclei  of  the  corpuscles,  both  red  and 
white,  are  stained  of  a brilliant  red,  although  the  surrounding  part 
of  the  corpuscle  is  not  so  stained,  unless  the  magenta  be  in  great 
excess.  All  the  corpuscles  are  not  stained  equally  brightly.  On 
the  edge  of  some  of  the  corjmscles  at  one  or  more  points  will  be 
found  small  coloured  spots  or  thickenings. 


Fig 

on  the  Red  Blood-Corpuscles 
of  Man  (a,  h)  and  Frog  (c). 


Fig.  67.— Action  of  Boracic  Acid  on  a Fi-og’s 
Red  Blood-Corpuscle. 


no 


PRACTICAL  HISTOLOGY. 


[II. 


9.  Osmic  Acid  and  Picrocarmine  (H). — This  preparation  is 
best  made  by  mixing  a few  drops  of  blood  with  an  equal  volume  of 
I per  cent,  osmic  acid  in  a small  tightly-corked  tube,  or  by  exposing 
a thin  him  of  blood  to  the  vapour  of  a 2 per  cent,  solution  of  osmic 
acid.  After  two  to  four  hours  pour  off  the  supernatant  fluid  and 
cover  the  residue  of-  corpuscles  with  picrocarmine.  After  twenty- 
four  hours  the  picrocarmine  can  be  poured  off,  and  a little  of  the 
deposit  placed  on  a slide  and  mixed  with  glycerine-jelly  dissolved 
by  heat,  and  covered  (p.  85). 

(a.)  Observe  that  the  nuclei  of  the  corpuscles  are  bright-red,  and 
the  perinuclear  part  of  the  corpuscles  yellow.  Within  the  granular- 
looking  nucleus,  with  a good  lens,  may  be  seen  a network  of  fibrils. 

This  preparation  is  permanent,  and  must  be  sealed  up  or  “ ringed 
after  the  manner  described  at  p.  88. 

10.  Blood  of  Bird  (H).  — Mount  a drop  of  fresh  blood. 

(a.)  Observe  the  red  corpuscles,  which  are  elliptical,  biconvex, 
nucleated  bodies,  but  ^smaller  in  size  and  more  pointed  than 
amphibian  corpuscles  (fig.  68). 

These  corpuscles  behave  towards  reagents  as  those  of  amphibians. 

11.  Blood  of  Fish  (H).  - — Mount  a drop  of  fresh  blood,  which  is 
readily  obtained  from  a gold-fish  or  salmon,  but  the  blood  coagulates 
rapidly.  ^ 

(a.)  Observe  the  red  corpuscles,  which  are  elliptical,  biconvex, 
and  nucleated,  but  the  ends  are  not  so  pointed  as  in  the  bird,  while, 

lil^  e the  bird’s  corpuscles, 
they  are  smaller  than 
those  of  amphibians  (fig. 
68,  F).  The  blood  must 
be  quite  fresh. 

In  both  cases  a few 
colourless  corpuscles  may 
be  noticed. 

(B.)  The  White  Cor- 
puscles of  the  Frog  or 
Newt. 

12.  White  Corpuscles. — In  a fresh  preparation  of  blood  (taking 
care  to  place  a hair  under  the  cover- glass)  search  for  the  colourless 
corpuscles,  of  which  there  are  several  varieties.  They  are  much 
less  numerous  than  the  red. 

(a.)  Observe  that  there  is:  (i.)  The  finely  granular  form,  con- 
sisting of  a nucleated  mass  of  protoplasm  larger  than  a red  corpuscle 
(fig.  61,  m).  (ii.)  The  coarsely  granular  variety  may  be  found.  In 

it  the  granules  are  large  and  refractive,  and  often  lying  at  one  side 
of  the  corpuscles,  (iii.)  A third  variety,  much  smaller  than  the 
others,  may  be  found  (fig.  61,  k). 


Fig.  68. — Red  Blood-Corpuscles  of  Fish  (F)  and 
Bird  (B),  x 450. 


II.] 


THE  BLOOD. 


Ill 


(b.)  In  all  the  varieties,  by  careful  observation,  may  be  detected 
a nucleus,  which  in  some  is  irregular  or  subdivided.  The  surface  of 
these  corpuscles  is  sticky,  as  can  readily  be  shown  by  giving  the 
cover  a push  with  a needle,  when  the  coloured  corpuscles  will  be 
seen  to  glide  over  each  other,  while  some  of  the  colourless  ones  will 
be  seen  adhering  to  the  glass ; and  even  if  a coloured  one  impinges 
on  them,  they  are  rarely  displaced  by  the  impact,  so  firmly  do  they 
adhere  to  the  glass. 

13.  Amoeboid  Movements  of  the  White  Corpuscles. — Select 
one  of  the  large  finely  granular  corpuscles,  and  at  once  make  a 
sketch  of  its  outline ; 
in  a minute  or  two 
make  another  sketch, 
and  do  this  every  few 
ininutes.  The  sketches 
will  vary,  because  the  fig.  69.- Showing  the  Ameboid 
corpuscle  has  slowly 

changed  its  shape,  even  at  the  ordinary  temperature.  A process  of 
its  protoplasm  may  be  extruded  on  one  side,  while  a part  of  the 
corpuscle  may  be  drawn  in  at  another.  The  corpuscle,  therefore, 
exhibits  spontaneous  irregular  and  indefinite  movements^  constitut- 
ing what  is  known  as  amoeboid  movements.  It  may  even  be  seen  to 
change  its  place,  and  thus  exhibit  locomotion. 

In  the  coarsely  granular  form,  if  one  be  found,  the  processes  are 
not  so  pointed,  while  the  granules  may  be  seen  to  pass  suddenly 


Fig.  70. — Banvier’s  Moist  Chamber,  s.  Disc  on  which  the  fluid  to  be  examined  is 
placed  ; d.  Air-Chamber ; c.  should  be  placed  on  the  square  part  on  which  the 
cover-glass  rests. 

from  one  side  of  the  corpuscle  to  the  other.  The  granules  seem  to 
be  passive,  and  their  motion  is  due  to  movements  of  the  protoplasm. 

These  movements  may  be  watched  for  a long  time  if  the  pre- 
paration be  sealed  up  either  with  melted  paraffin  wax  or  with  oil. 
The  former  is  to  be  preferred.  When  sealed  up  in  paraffin  wax,  the 
preparation  may  be  kept  without  evaporation  for  several  days.  The 
margins  of  the  cover-glass — preferably  a square  one — are  sealed  down 


PRACTICAL  HISTOLOGY. 


II2 


[II. 


with  paraffin  wax  by  heating  a wire  bent  at  right  angles  to  melt 
the  paraffin  and  applying  it  along  the  edges  of  the  cover-glass. 

Eanvier  uses  a moist  air-chamber  (fig.  70).  In  the  centre  of  the 
chamber  is  a small  piece  of  glass,  less  in  height  by  y\^th  of  a milli- 
metre than  the  height  of  the  walls  of  the  cavity.  The  fluid  con- 
taining the  corpuscles  is  placed  on  this,  covered  with  a cover-glass 
which  is  sealed  down  by  means  of  paraffin  wax.  The  object  is  thus 
protected  from  evaporation,  while  it  has  in  relation  to  it  a layer 
of  air. 

14.  Acetic  Acid  (i  per  cent.). — On  irrigating  a fresh  preparation 
with  dilute  acetic  acid,  the  protoplasm  is  made  clear  and  transparent, 

and  the  complex  nucleus — 
usually  consisting  of  three 
parts,  and  hence  called  tri- 
partite — is  distinctly  re- 
vealed (fig.  71,  a).  If  the 
acid  act  vigorously,  it  may 
be  almost  impossible  to  see 
the  outline  of  the  now  clear 
protoplasm. 

15.  Water. — On  irrigat- 
ing a fresh  preparation  with  water,  the  colourless  corpuscles  are 
killed,  and  they  assume  a globular  form,  the  protoplasm  becoming 
at  first  more  granular  (fig.  71,  Z>),  and  subsequently  clear  and  trans- 
parent, thus  distinctly  revealing  the  presence  of  the  nucleus.  The 
granules  may  exhibit  Brownian  movements,  while  each  cell  has  an 
outline  round  it  as  if  a membrane  had  been  formed  round  it.  So 
that  by  the  addition  of  water  these  corpuscles  may  be  changed  so  as 
to  resemble  salivary  corpuscles  (Lesson  lY.). 

16.  Magenta. — This  stains  the  nucleus  deeply  and  the  proto- 
plasm to  a less  degree. 

For  Fibrin  (Lesson  III. 


FlO.  71. — a.  Action  of  Acetic  Acid  on  the  Colourless 
Blood-Corpuscles  of  a Frog.  Action  of  Water 
on  the  Colourless  Blood-Corpuscles  of  a Frog. 
h.  Early,  and  c.  Later  Stages. 


Lymph. 

17.  {a.)  Pith  a frog,  and  with  a fine  pipette  withdraw  a little 
lymph  from  the  dorsal  lymph  sac,  or,  better  still,  curarise  a pithed 
frog,  and  next  day  remove  the  lymph  from  its  sub-lingual  lymph 
sac.  The  lymph  accumulates  there  in  large  amount. 

Lymph  (H). 

(a.)  Observe  numerous  leucocytes,  mixed  perhaps  with  a few  red 
blood-corpuscles,  exactly  like  the  three  varieties  of  leucocytes  found 
in  the  blood.  A coagulum  of  fibrin  will  ultimately  be  formed. 

(b.)  The  toad  yields  a very  large  quantity  of  lymph.  Destroy 
its  brain,  wipe  one  leg  dry,  and  cut  off  the  projecting  toe.  At  once 
large  drops  of  lymph  flow  out,  which  soon  coagulates  {S,  Mayer), 


nl 


THE  BLOOD. 


1 13 


ADDITIO^^AL  EXERCISES. 

18.  Migration  of  Colourless  Corpuscles  ( H ).  — Heat  and  draw  out  a glass 
tube  so  as  to  form  an  excessively  fine  capillary  tube.  Pith  a frog,  expose  its 
heart,  make  a cut  into  the  latter,  and  as  the  blood  flows  suck  up  blood  into  the 
capillary  tube.  Seal  the  tube  at  both  ends  by  holding  it  for  a second  or  two 
in  a gas-flame. 

Place  the  tube  on  a slide  in  a drop  of  glycerine  or  clove-oil,  cover  it  and 
examine. 

(^•)  (L)  Observe  that  the  blood-clot  shrinks,  squeezes  out  serum  from  the 
clot,  and  b}'-and-by  some  of  the  colourless  corpuscles  migrate  from  or  are 
s(]ueezed  out  of  the  red  clot  into  the  serum,  where  they  may  be  seen  exhibiting 
amoeboid  movements.  This  shows  that  the  vitality  of  the  white  corpuscles  is 
not  abolished  by  the  coagulation  of  the  blood. 

19.  Feeding  White  Corpuscles  (H ). — Get  some  blood  from  the  heart  of  a 
frog  into  a capillary  pipette,  as  described  in  18.  Allow  the  blood  to  clot  and 
exude  serum,  whicli  it  does  in  about  an  hour. 

Rub  up  a iittle  Indian -ink  in  a few  drops  of  normal  saline  in  a v/atch-glass 
till  a greyish  fluid  is  obtained.  Blow  the  contents  of  the  capillary  tube  upon 
a clean  slide,  remove  the  clot ; the  serum  contains  numerous  colourless  cor- 
puscles. ^lix  a little  of  the  greyish  Indian-ink  fluid  with  the  serum,  put 
a liair  under  the  cover-glass,  and  seal  up  the  latter  with  melted  ]»araffin  to 
prevent  evaporation.  After  a time,  observe  that  the  minute  particles  of 
Indian-ink  are  seen  included  in  the  protoplasm  of  these  cells.  The  cells  throw 
out  processes  which  surround  a particle,  meet  and  coalesce,  and  thus  the 
particles  come  to  be  included  in  the  corpuscle  {Schafer). 

20.  Movements  and  Division  of  White  Corpuscles. — Ranvier^  seals  up  the 
preparation  on  the  slide  devised  by  him,  this  being  done  by  paraffin  wax.  A 
water  immersion  lens  is  placed  on  the  microscope,  and  then  the  microscope 
with  the  slide  is  placed  in  a glass  vessel  filled  with  water  at  the  required 
temperature.  In  this  way  the  movements  of  the  corpuscles,  and,  as  I have 
myself  seen,  even  the  division  of  leucocytes,  can  be  readily  studied. 

21.  Glycogen  in  White  Corpuscles  ( H ).  — Irrigate  a preparation  of  frog’s 
or  newt’s  blood  with  I per  cent,  solution  of  iodine  containing  2 grams  of 
potassic  iodide.  The  red  corpuscles  are  stained  yellow,  the  white  ones  are 
killed  ; many  of  them  are  also  stained  yellow,  but  in  some  of  them  may  be 
seen  mahogany-coloured  granules  of  stained  glycogen. 

22.  Elder  Pith  Preparation  (H). — Into  the  dorsal  sac  of  a pithed  frog 
introduce  a small  piece  of  elder  pith  soaked  in  normal  saline,  and  leave  it 
there  fi»r  twenty-four  hours.  Withdraw  it,  make  a thin  section,  and  examine 
it  in  normal  saline  solution. 

{a. ) Observe  the  large  polygonal  cells  of  the  elder  pith  crowded  with  lymph 
corpuscles — several  varieties — which  have,  in  virtue  of  their  amoeboid  move- 
ments, “wandered”  into  and  permeated  the  cellular  pith. 

The  elder  pith  may  be  hardened  in  Flemming’s  fluid  (p.  32),  and  after 
thoroughly  washing  it,  sections  are  made  and  mounted  in  Tarrant’s  solution. 

23.  Serous  Fluids  of  Mammals.— A small  mammal,  c.g.,  mouse,  rabbit,  is 
killed  by  decapitation.  Open  its  peritoneal  cavity  by  means  of  a sharp  red-hot 
knife,  Lc.,  by  a thermo-cautery.  This  is  to  avoid  any  blood  being  shed  into 
the  cavity.  By  means  of  a fine  ]>ipette  withdraw  some  of  the  “lymph”  or 
“ serosity.”  It  will  be  found  not  to  be  clear  like  whey,  but  slightly  opalescent. 

{a.)  Corpuscles  (H). — Some  are  like  ordinary  lymph  corpuscles,  many  are 
very  granular,  and  there  are  always  red  blood-corpuscles  also  present  {Eanvier).'^ 

^ Comptes  Hendus,  vol.  110,  p.  686,  1890. 

2 Ibid.  vol.  110,  p.  768,  1890. 


H 


PRACTICAL  HISTOLOGY. 


114 


[II. 


24.  Dry  Cover-Glass  Preparation  of  Blood. — Place  a drop  of  frog’s  blood 
on  a cover*glass,  and  to  it  apply  another  cover-glass.  Press  the  two  glasses 
together,  and  tlien  slip  them  asunder.  There  will  be  a thin  film  of  blood  on 
both  glasses.  Allow  the  films  to  dry. 

In  a watch-glass  place  a dilute  watery  solution  of  methyl-green,  and  on  this 
float  the  cover-glasses,  with  the  blood-surface  next  the  staining  reagent,  as  in 
the  method  of  staining  bacteria. 

After  ten  minutes  remove  the  cover-glass  ; move  it  in  water  ; touch  the 
edge  of  it  on  blotting-paper  to  remove  the  surplus  green  fluid,  and  allow  the 
green  stain  on  the  glass  to  dry.  After  it  is  dry,  add  a drop  of  xylol  balsam 
and  place  the  cover-glass  on  a slide.  This  forms  a permanent  preparation  of 
blood-corpuscles,  whose  nuclei  are  stained  green. 

The  same  process  may  be  practised  with  other  aniline  dyes,  or  eosin  may  be 
combined  with  methyl-green,  as  the  former  stains  the  haemoglobin,  and  the 
latter  the  nucleus.  A very  good  stain  is  eosin-haematoxylin  (p.  70). 

25.  Double-Staining  of  Blood- Corpuscles  (Methyl-green  arid  Eosin).  — Diffuse 
a thin  layer  of  frog’s  blood  on  a cover-glass,  allow  it  to  dry,  and  pour  on 
the  dry  residue  a i per  cent,  watery  solution  of  methyl-green.  Leave  it  on 
for  ten  minutes,  and  then  wash  off  the  surplus  stain.  Pour  on  a weak  watery 
solution  of  eosin,  and  after  five  minutes  wash  it  off  also  by  rinsing  the  slide 
gently  in  water  ; dry,  and  add  xylol  balsam  ; cover. 

Observe  that  some  of  the  corpuscles  have  the  nucleus  green  and  the  sur- 
rounding part  coppery-red.  Eosin  is  almost  a specific  reagent  for  detecting 
haemoglobin. 

Numerous  combinations  of  this  kind  may  be  made,  such  as  magenta  and 
iodine-green,  fuchsin  and  methylene-blue. 

26.  A better  plan  is  to  place  a drop  of  blood  on  a cover-glass,  and  to  this 
apply  another  cover-glass,  press  the  glasses  together,  and  then  separate  them 
so  that  a thin  film  of  blood  adheres  to  each.  Allow  them  to  dry.  After  they 
are  dry,  place  them  for  two  hours  in  a mixture  of  equal  parts  of  absolute 
alcohol  and  ether,  which  coagulates  the  proteids  of  the  corpuscles.  Float 
the  cover-glasses  blood-surface  downwards  upon  a saturated  solution  of  methy- 
lene-blue. After  an  hour  or  more,  wash  the  cover-glasses  in  water,  then  in 
absolute  alcohol,  and  clear  them  up  with  clove-oil  in  which  a little  eosin  is 
dissolved.  Remove  the  clove-oil  by  immersing  the  cover-glasses  in  xylol,  and 
mount  in  xylol  balsam.  A beautiful  preparation  is  obtained.  The  nuclei  are 
blue,  and  the  protoplasm  of  the  colourless  corpuscles  of  pale-rose  colour. 

27.  The  advanced  student  should  also  stinly  the  effect  of  sulpho-cyanide 
of  potassium  (5  per  cent.),  urea,  ammonium  chromate,  pyrogallic  acid,  heat, 
carbolic  acid  (i  to  1000  of  normal  saline),  and  other  agents  on  the  coloured 
corpuscles.  Dilute  alcohol  reveals  a nucleolus  within  the  nucleus.  This 
pre])aration  may  be  subsequently  stained  with  magenta. 

28.  Urea.  — A strong  watery  solution  causes  the  corpuscles  to  assume 
irregular  forms  and  to  send  out  processes,  which  may  separate  from  the 
corpuscle.  Changes  not  unlike  these  are  produced  by  neutral  ammonium 
chromate,  very  bizarre  forms  being  thus  ]»roduced. 

29.  Bile,  e.g.,  of  mammal  or  frog,  dissolves  human  red  blood-corpuscles. 

30.  Zinc  Sulphate  (.25-. 5 per  cent.)  causes  the  h?emoglobin  to  separate 
from  the  stroma.  One  may  see  it  inside  the  corpuscle,  or  on  the  surface  as 
small  buds. 

31.  Pseudo-Membrane  of  the  Corpuscles. — Irrigate  a preparation  with 
dilute  alcohol.  This  decolorises  the  corpuscles.  Then  stain  with  magenta 
or  Spider’s  purple.  The  nucleus  and  so-called  membrane  of  the  corpuscles  are 
thereby  stained. 

32.  Preservation  of  Blood-Corpuscles  by  Hayem’s  Fluid  (p.  122). — This  is 
an  excellent  method.  The  corpuscles  retain  their  form,  and  can  be  subsequently 


III.] 


HUMAN  BLOOD. 


II? 

Rfc^iiied,  e.g.^  by  picro-carmine,  and  kee[>  well  when  mounted  in  glycerine 
jelly. 

33.  Leucocytes  of  Crayfish  Blood,  or  Hsemolymph. — The  colourless  blood  of 
the  crayfish  does  very  well  for  the  study  of  colourless  corpuscles.  Make  a slit 
in  the  ventral  surface  of  the  abdomen  between  the  rings  and  the  blood  flows 
freely.  Beceive  it  in  normal  saline  solution.  It  clots  quickly.  Mount  some  on 
a slide,  and  if  it  be  desired  to  fix  the  corjmscles,  allow  a jet  of  steam  to  }day  on 
the  cover-glass  for  a few  seconds.  If  the  fluid  contain  too  much  blood  there  is 
so  much  coagulable  proteid,  that  on  its  coagulation  by  the  steam  a white  film 
obscuring  the  leucocytes  is  formed.  The  corpuscles  can  be  subsequently  stained 
with  picro-carmine  and  mounted  in  glycerine.  The  blood  contains  two  forms 
of  corpuscles — one  with  well-maiked  amceboid  movements  and  provided  with  a 
large  spherical  nucleus  ; the  other  filled  with  highly  retractile  granules,  which 
stain  red  with  picro-carmine. 

The  best  method  is  to  allow  a drop  of  blood  to  fall  into  a large  drop  of  i , 
per  cent,  osmic  acid  pieviously  placed  on  a slide.  This  at  once  kills  and 
“fixes  ” the  corpuscles,  which  can  then  be  stained  with  picro-carmine,  or  i per 
cent,  watery  solution  of  eosin.  The  latter  stains  their  protoplasma  and  its 
expansions  a rose-pink. 


LESSON  III. 

HUMAN  BLOOD— CRYSTALS  FROM  BLOOD- 
BLOOD  PLATELETS. 

Wrap  a twisted  handkerchief  round  the  ring-finger  of  the  left  liand, 
and  begin  at  the  base  of  the  finger,  gradually  constricting  the 
finger  from  the  base  towards  the  nail.  The  end  phalanx  will  there- 
by become  greatly  congested.  With  a sharp  clean  sewing-needle 
prick  the  skin  at  the  root  of  the  nail ; a drop  of  blood  will  exude, 
to  which  rapidly  apply  a slide  ; cover  the  drop  of  blood  on  the  slide 
with  a cover-glass,  and  examine  it  as  quickly  as  possible. 

Observe,  various  kinds  of  corpuscles  floating  in  a fluid,  the 
blood-plasma  or  liquor  sanguinis.  Note  the  red  and  white  cor- 
puscles, the  former  being  far  more  numerous.  Blood  platelets  are 
also  present ; but  it  requires  special  precautions  in  order  to  preserve 
them. 

1.  Human  Coloured  Blood-Corpuscles  (H). — (a.)  Observe  the 
field  of  the  microscope  crowded  with  the  red  or  coloured  disc-shaped 
corpuscles  much  smaller  than  those  of  the  newt ; they  are  only 
3 2Vo^^^  of  an  inch  or  7.7  /x  (7.2  /x-7.8  /x)  in  breadth  (p.  21),  and 
or  2 /X  in  thickness.  The  observer  may  notice 
that  when  the  corpuscles  cease  to  move,  after  a time  corpuscles  may 
be  seen  adhering  to  each  other  by  their  flat  surfaces,  until  a chain 


PRACTICAL  HISTOLOGY. 


[HI. 


1 16 


Pig.  72. — Human  Red  and  White  Blood-Corpuscles. 
a.  Red  corpuscle  seen  on  the  flat ; h.  In  profile; 
c,  Arouleau;  d.  Three-quarter  face;  e,f.  Crenated 
corpuscles  ; g.  Spherical ; m.  Slightl}'  crenated  ; 
L.  Large  white  corpuscle ; 1.  Small  white  cor- 
puscle ; p.  Granular  leucocyte ; n.  Pree  granula- 
tions, X 1000. 


of  these  bodies  resembling  a pile  of  coins  is  produced  (fig.  72). 
This  is  the  so-called  formation  of  rouleaux  (fig.  72,  c).  In  order  to 

obtain  rouleaux  a fairly 
large  drop  of  blood  must 
be  taken.  These  chains  by- 
and-by  increase  in  number 
and  intersect  each  other,  so 
as  to  produce  a network-like 
appearance  in  the  field.  In 
the  rouleaux  all  the  cor- 
puscles are  seen  edgeways. 

{h.)  Move  the  preparation 
until  a part  of  it  is  found 
where  the  red  corpuscles 
are  to  be  seen  not  in 
rouleaux^  but  isolated  and 
13/ing  on  the  flat. 

Study  a Single  Eed  Cor- 
puscle.— Observe  its  shape  ; when  seen  on  the  flat  it  is  circular  in 
outline,  and  on  bringing  its  edge  sharply  into  focus,  a darker  area 
is  seen  in  its  centre  (fig.  74,  a).  If  the  fine 
adjustment  be  used,  so  as  to  bring  the  lens 
nearer  the  corpuscle,  the  dark  centre  is  replaced 
by  a lighter  area,  while  the  rim  becomes  darker 

(fig-  74.  ^)- 

Sketch  the  two  appearances. 

This  is  due  to  the  fact  that  these  bodies 
are  hi-concave  circular  discs.  The  dark  area 
in  the  centre  is  not  due  to  the  presence  of  a 
nucleus,  as  they  are  non-nucleated.^ 

This  is  confirmed  by  examining  a corpuscle 
seen  on  edge,  when  it  appears  somewhat  dumb- 
bell shaped  (figs.  72,  h,  73,  2). 

In  size  they  are  much  smaller  than  those  of  Amphibia,  being  only 
of  an  inch  (7.7  p)  in  their  greatest  diameter.  Their  border 
is  smooth,  rounded,  and  regular,  their  colour  is 
^ ^ pale  straw-yellow,  their  surfaces  are  smooth,  and 

tliey  are  homogeneous  throughout.  As  to  trans- 
parency^ the  outline  of  one  corpuscle  can  be  seen 
through  one  overlapping  it.  They  are  soft  and 
flexible,  so  that  if  they  impinge  on  other  objects 
they  may  change  their  form,  which  they  rapidly 
regain,  so  that  they  are  elastic. 

(c.)  Measure  the  actual  size  of  several  corpuscles 
by  the  method  described  at  p.  20.  It  will  be  found  that  all  the 


Fig.  73.— Red  Blood-Cor- 
puscles. Human  ; i. 
Seen  on  their  surface ; 
2.  Seen  edgeways ; 3. 
United  into  a rouleau. 


Fig.  74.— Red  Cor- 
puscle seen  on  the 
flat.  a.  On  rais- 
ing, and  h.  On 
depressing  the  ob- 
jective, X 1000. 


III.] 


HUMAN  BLOOD. 


II7 

corpuscles  are  not  of  absolutely  the  same  size.  This  variation  in 
size  becomes  more  marked  in  some  diseases. 

2.  Colourless  Corpuscles. — With  a little  care  in  observing, 
especially  when  the  red  corpuscles  are  in  rouleaux^  here  and  there  in 
the  Held  a few  colourless  corpuscles  will  be  found.  They  remain 
isolated  and  do  not  form  groups.  They  are  few  in  iiumber,  only 
three  to  ten  being  found  in  one  field.  The  proportion  is  about  3 to 
10  per  1000  of  red.  They  are  usually  spherical,  and  although  they 
exhibit  amoeboid  movements  at  40°  C.,  at  the  ordinary  temperature 
they  do  not  do  so,  and  appear  as  nucleated  finely  granular  masses  of 
protoplasm.  They  rapidly  alter  after  they  are  shed.  Some  of  them 
are  larger  (-o/w  or  10  /x  in  diameter),  others  smaller  than  the 
red  corpuscles  (fig.  72,  L,  T). 

Move  the  cover-glass  so  as  to  cause  a current  in  the  preparation. 
Note  that  the  colourless  corpuscles  adhere  to  the  glass, — they  are 
sticky  and  adhesive, — while  the  coloured  ones  being  smooth,  and 
polished,  glide  over  each  other,  and  frequently  impinge  on  the 
colourless  ones  without  displacing  the  latter. 

3.  Blood-Plates  (see  p.  123). 

4.  Acetic  Acid,  Tannic  Acid  (fig.  66,  a,  Z/),  Water,  Syrup,  and 
Magenta  act  in  great  part  in  the  same  manner  on  the  coloured  and 
colourless  corpuscles  of  man  and  mammalia  as  on  the  corpuscles  of 
Amphibia.  The  differences  are  due  to  structural  differences  in  the 
corpuscles. 

Thus  in  the  coloured  mammalian  corpuscles  acetic  acid  de- 
colorises them  and  renders  them  spherical  (but  of  course  no  nucleus 
is  revealed),  leaving  a hull  or  stroma,  almost  invisible  in  the  field. 
Syrup  shrivels  them,  magenta  reveals  no  nucleus,  but  on  the  side 
of  some  of  the  corpuscles  a little  spot  may  be  observed.  Bile  makes 
them  pale,  and  finally  dissolves  them.  Water  decolorises  the  red 
corpuscles,  and  makes  the  blood  ‘‘laky.  ” 

5.  Amoeboid  Movements  of  Wliite  Blood-Corpuscles. — In  order 
to  study  these  movements  in  the  white  blood-corpuscles  of  man  and 
warm-blooded  animals  generally,  the  temperature  of  the  blood  must 
bo  near  the  temperature  of  the  body.  To  keep  the  preparation 
warm,  some  form  of  warm  or  hot  stage  is  necessary. 

For  the  purposes  of  the  student  the  following  simple  contrivance 
is  sufficient.  Take  an  oblong  copper  plate,  76  mm.  long,  25  mm. 
broad,  and  1.5  mm.  thick,  with  a rod  100  mm.  long  projecting  from 
one  side  of  it,  as  shown  in  fig.  75.  In  the  centre  of  the  oblong 
plate  of  copper  is  a hole  15  mm.  in  diameter.  Fix  the  oblong  plate 
to  an  ordinary  glass  slide  by  means  of  sealing-wax. 

Make  a preparation  of  blood.  Take  a cover-glass  i inch  square, 
and  on  it  place  a drop  of  normal  saline  solution,  and  to  the  latter 
add  a droj)  of  blood.  Mix  them.  Apply  a |-inch  cover-glass,  so  as 


ii8 


PRACTICAL  HISTOLOGY. 


[III. 


to  have  a layer  of  diluted  blood  in  a thin  film  between  the  two 
cover-glasses,  and ' exactly  filling  the  space  between  them.  Any 
surplus  fluid  at  the  edge  of  the  cover- 
glasses  must  be  removed  by  blotting-paper. 
With  a camel’s-hair  pencil  then  run  a layer 
of  oil  round  the  edge  of  the  smaller  cover- 
glass,  or  the  preparation  may  be  sealed  up 
with  melted  paraffin  wax.  This  will  pre- 
vent evaporation.  The  larger  cover-glass 
acts  the  part  of  a slide. 

Fix  the  warm  stage  on  the  stage  of  the 
microscope  by  means  of  the  clips  on  the 
microscope,  and  over  the  aperture  in  the 
warm  stage  place  the  cover-glasses.  Focus 
the  corpuscle  between  the  two  cover- 
glasses,  and  notice  that  the  colourless 
ones  do  not  exhibit  amoeboid  move- 
ments. 

Heat  the  projecting  rod  by  means  of  a 
spirit-lamp.  The  heat  travels  along  the 
copper,  and  finally  warms  the  cover-glasses 
and  the  layer  of  blood  between  them.  It 
must,  however,  not  be  overheated.  In 
order  to  prevent  overheating,  make  pre- 
viously a mixture  of  cacao-butter  and 
white  wax,  which  melts  at  38°  C.,  and 
place  a fragment  of  this  on  the  copper 
stem  near  the  copper  plate.  Heat  should 
be  applied  until  the  wax  just  begins  to  melt. 

Observe  the  colourless  corpuscles  when  heated  beginning  to 


Fia.  75.— Simple  Copper  Warm 
Stage. 


Fig.  76. — Warm  Stage  made  by  Reichert  of  Vienna.  It  costs  £1.  A,  A'.  Screws  to  fix  it 
to  the  stage  of  a microscope  ; B.  Inflow,  and  B'.  Outflow  of  water. 


exhibit  amoeboid  movement.  Sketch  a corpuscle,  and  in  a few 
minutes  make  another  sketch  and  compare  the  two. 


III.]  HUMAN  BLOOD.  I I9 

In  the  more  expensive  forms  of  warm  stages,  as  those  of  M. 
Schultze  and  others,  warm  water  at  a known  temperature  is  passed 
through  a brass  box  which  rests  on  the  stage  of  the  microscope,  and 
the  exact  temperature  is  determined  by  means  of  a delicate  thermo- 
meter. Fig.  76  shows  a convenient  form  of  hot  stage,  which  can  be 
clamped  to  the  stage  of  any  microscope.  It  is  heated  by  means  of 
warm  water  which  passes  in  at  B,  and,  after  traversing  a system  of 
tubes,  out  at  B'.  It  is  ]wovided  with  a thermometer. 

6.  Crenation  of  Coloured  Corpuscles  (H). — Mix  a drop  of 
human  blood  with  a 2 per  cent,  solution  of  common  salt.  Note 
the  change  of  colour.  (a.)  Observe  that 
some  corpuscles  shrink  in  part,  and  become 
crenate  or  beset  with  short  spines  (fig.  77). 

This  is  *due  to  exosmosis  of  fluid  from  the 
corpuscles.  The  colour  becomes  slightly 
deeper  than  in  normal  corpuscles.  All  the 
corpuscles  are  not  affected  simultaneously 
or  to  the  same  extent.' 

In  some  individuals,  merely  exposing  the 
blood  to  the  air  for  a few  minutes  before 
applying  a cover-glass  suffices  to  produce 
this  condition  (fig.  72,  e,  f);  but  in  any 

specimen  of  blood  it  may  be  readily  produced  in  the  majority  of 
red  corpuscles  by  acting  on  them  with  a saline  solution  of  appro- 
priate concentration. 

It  has  been  observed  that  in  the  blood  of  a mammal  poisoned  by 
Calabar-bean  the  blood-corpuscles  are  crenated.  * 

7.  Dilute  Alkalies. — Use  a 0.2  per  cent,  solution  of  caustic 
potash  (i.e.,  2 grams  in  1000  cc.  of  normal  saline).  It.  dissolves 
both  the  red  and  white  corpuscles. 

8.  Fibrin  (H). — Make  a preparation  of  human  blood,  using  a 
large  drop.  Cover  it  and  put  it  aside  for  half -an-h our  or  longer 
until  the  blood  coagulates. 

(a.)  Observe  carefully,  and  in  the  meshes  between  the  rouleaux 
fine  threads  forming  a delicate  network  will  be  seen.  They  are 
fibrils  of  fibrin. 

{h.)  A better  method,  however,  is  to  mix  on  a slide  a drop  of 
blood  with  a drop  of  normal  saline  solution.  Cover  and  put  it 
aside  for  an  hour  or  so  to  clot,  and  then  irrigate  with  water  or  dilute 
alcohol  (p.  25),  which  rapidly  decolorises  and  washes  away  the  red 
corpuscles,  and  thus  brings  into  view  a fine  fibrillar  network  of  fibrin 
in  the  field.  Irrigate  with  a watery  solution  of  SpilleFs  purple  (i 
per  cent.),  which  stains  the  network  of  fibrin  a purplish  tinge  (fig. 
78).  Raise  the  cover-glass,  and  to  it  will  be  found  adhering  a thin 
film  of  fibrin.  Dry  the  film,  apply  a drop  of  balsam,  and  mount  the 


120 


PRACTICAL  HISTOLOGY. 


[III. 


specimen  as  a })ermaiient  preparation.  Numerous  purple  stained 
threads  of  fibrin  are  seen  stretching  from  colourless  corpuscles,  the 
latter  having  their  nuclei  stained  purple. 

Crystals  from  Blood. — The  haemoglobin  of  certain  animals 
crystallises  very  readily,  e.^.,  rat,  guinea-pig. 

9.  Haemoglobin  Crystals  of  B^t’s  Blood  (H). — Place  a drop  of 
defibrinated  rat’s  blood  on  a slide,  add  two  drops  of  water,  and  mix. 
Apply  a cover-glass.  After  a few  minutes,  near  the  edge  of  the 
cover-glass  oblique  rhombic  crystals  of  haemoglobin  will  be  found. 

‘ At  first  they  are  small,  but  they  gradually  become  larger.  They 
may  be  single,  or  arranged  in  rosettes,  or  crossing  each  other 
(fig-  79)- 

10.  Crystals  from  Guinea-Pig’s  Blood  (H). — To  a drop  of  the 


Fig.  78.— Fibrils  of  Fibrin 
of  Coagulated  Blood. 


Fig.  79. — Haemoglobin  Crystals  from 
Bat’s  Blood,  x 300. 


Fig.  80.— Haemo- 
globin Crystals 
from  Blood  of 
Guinea-Pig. 


defibrinated  blood  add  a drop  of  Canada  balsam  or  clove-oil,  mix, 
and  apply  a cover-glass.  It  is  perhaps  better  to  place  the  balsam 
or  clove-oil  on  the  slide  first,  then  to  place  the  drop  of  blood  on 
the  top  of  the  oil  or  balsam,  and  then  to  mix  them  ; very  soon 
(about  10  minutes)  large  red  tetrahedral  crystals  are  formed. 
These  crystals  cannot  be  preserved  for  any  length  of  time  (fig.  80). 

• Sometimes  very  good  crystals  of  haemoglobin  from  human  blood 
are  obtained  from  leeches  which  have  sucked  blood  some  weeks 
previously. 

11.  Hsemin  Crystals  (H). — Place  a few  particles  of  dried  blood 
on  a slide,  add  a small  crystal  of  common  salt,  and  two  drops  of 
glacial  acetic  acid.  Cover.  Heat  over  the  flame  of  a spirit-lamp 
until  bubbles  of  gas  are  given  off,  i.e.^  until  it  boils.  Allow  it  to 
cool.  Or  mix  fresh  blood  on  a slide  with  a minute  quantity  of  2 per 


III.] 


CRYSTALS  FROM  BLOOD. 


I2I 


cent,  salt  solution.  Heat  tlie  mixture  until  it  becomes  brownish  in 
tint,  add  glacial  acetic  acid  and  heat.  On  cooling,  crystals  of  haemin 
are  obtained.  Should  there  be  any  cubes  of  common  salt  present 
irrigate  with  water,  which  soon  removes  the  latter.  Haemin  crystals 
are  insoluble  in  water. 

(a.)  Observe  the  small  brownish  or  black  rhombic  crystals,  either 
singly  or  in  rosettes,  scattered  over  the  field  or  on  the  surface  of 
the  larger  masses  of  blood  (fig.  8i). 


(6.)  To  preserve  them,  remove  the  acid,  raise  the  cover-glass, 
dry  the  preparation,  and  mount  them  in  balsam. 

12.  Enumeration  of  the  Blood  Corpuscles. — See  the  author’s 
Outlines  of  Practical  Fhysiolofjy^  Lesson  YI.,  p.  42. 

13.  Leukaemic  Blood  (H). — If  a small  quantity  can  be  obtained 
from  a patient  in  the  hospital,  examine  it. 

(a.)  Observe  the  great  excess  of  colourless  corpuscles.  Accord- 
ing to  the  variety  of  the  leukaemia,  they  may  be  larger  or  smaller 
in  size  than  most  of  the  coloured  corpuscles  (fig.  82). 


ADDITIONAL  EXERCISP:S. 

14.  Human  Blood  and  Aniline  Dyes  (H;.— Mnke  a cover-glass  preparation 
of  human  blood  (Lesson  II.  24).  Ehrlich  heated  the  cover-glass  in  an  air- 
oven  to  a temperature  of  120°  for  several  hours  to  coagulate  the  proteids.  To 
avoid  this,  place  the  covers  for  two  hours  in  the  alcohol  and  ether  mixture 
(Lesson  II.  26),  and  then  stain  them  in  methylene-blue  as  directed  for  frog’s 
blood-corpuscles,  or  stain  in  i ]*er  cent.  Spider’s  purple  or  a weak  alcoholic 
solution  of  rosein.  After  washing  and  drying,  mount  them  in  xylol  balsam. 

In  the  methylene-blue  preparation  the  coloured  corpuscles  are  unstained, 
but  some  of  the  colourless  ones  liave  their  nuclei  stained  blue,  while  the 
surrounding  protoplasm  is  unaffected  ; in  other  colourless  corpuscles  the 
gi-anules  in  the  protoplasm  are  stained.  These  corpuscles  correspond  to 
Ehrlich’s  basophile  corpuscles.  In  the  other  preparations  both  the  coloured 
and  colourless  corpuscles  are  stained. 

12 


122 


PRACTICAL  HISTOLOGY. 


[III. 


15.  Varieties  of  Leucocytes  in  Blood. — According  to  Ehrlich,  the  following 
varieties  of  colourless  corpuscles  are  present  in  the  blood.  (Also  Lesson 
XXXVIII.) 

{a.)  Small  lymphocytes.  They  are  slightly  smaller  than  the  red  corpuscles, 
and  possess  a large  s[)liericnl  readily-stained  nucleus,  which  almost  tills  the 
cell,  being  surrounded  only  by  a small  quantity  of  protoplasm. 

{h.)  Large  lymphocytes  are  said  to  represent  an  advanced  stage  of  {a).  They 
are  twice  as  large  as  (a),  have  a large  nucleus  surrounded  by  a well-defined  zone 
of  protoplasm,  {a)  and  {h)  together  make  up  25  per  cent,  of  the  leucocytes 
in  blood. 

(c.)  Mononuclear  elements,  or  transition  forms,  are  distinguished  from  the 
large  lymphocytes  by  their  nucleus  not  being  quite  spherical,  and  having  a 
depression  in  the  middle. 

{d.)  Polynuclear  leucocytes,  which  are  smaller  than  (c),  but  larger  than 
red  blood -corpuscles.  They  contain  a nucleus  composed  of  several  lobes,  or 
several  nuclei  which  stain  readily  and  deeply.  They  represent  70  per  cent,  of 
all  the  leucocytes  of  the  blood,  and  can  migrate  from  the  vessels. 

(e. ) Eosinophilous  cells.  The  nucleus  stains  less  deeply  than  {d).  The 
granules  which  are  present  in  the  protoplasm  stain  deeply  with  eosin,  i.e., 
become  intensely  red.  They  occur  but  sparsely  in  normal  blood. 

16.  Staining  of  Leucocytes  (Ehrlich), 

(1.)  Make  a cover-glass  preparation  of  blood  and  dry  it  for  several  hours 
at  120°  C. 

(2.)  Stain  it  for  several  hours  in  Ehrlich’s  acid-hsematoxylin,  eosin 
solution,  or  in  a strong  glycerine  solution  of  eosin. 

(3.)  Wash  in  water, — dry  and  mount  in  xylol-balsam. 

The  nuclei  of  the  white  blood-corpuscles,  as  well  as  the  lymphocytes  and 
polynuclear  forms,  are  deeply  coloured  ; the  nuclei  of  the  mononuclear  forms 
are  bluish-gray,  the  red  corpuscles  copper-red,  and  the  eosinophile  granules 
red  (Kahlden). 

17.  Eosinophilous  Cells. — If  it  be  desired  to  stain  only  these  cells,  stain  a 
cover-glass  preparation  with  a strong  glycerine  solution  of  eosin.  The  results 
of  Ehrlich  and  his  pupils,  will  be  found  in  his  pamphlet^ 

18.  Action  of  Hayem’s  Fluid. — This  is  an  extremely  useful  fluid  for  pre- 
serving and  fixing  the  blood-corpuscles,  and  can  be  employed  both  for  the 
blood  of  animals  and  of  man.  It  consists  of 


Sodic  chloride  . 

Sodic  sulphate 
Corrosive  sublimate  . 
Distilled  water 


I grm. 
5 grms. 
0.5  grm. 
200  cc. 


The  blood  is  run  direct  from  a blood-vessel  into  this  fluid  in  the  proportion 
of  I of  blood  to  100  of  the  fluid.  It  takes  several  hours  to  harden  and  fix  the 
corpuscles,  but  twenty-four  hours  is  not  too  long.  By-and-by  the  corpuscles 
subside,  and  the  supernatant  fluid  can  then  be  decanted,  and  the  deposit  of 
blood-corpuscles  well  washed  with  water  to  get  rid  of  the  salts  of  the  mixture. 
These  corpuscles  can  then  be  stained  with  various  reagents,  or  eosin  may  be 
added  to  the  fluid. 

(a.)  Make  an  experiment  with  frog’s  blood,  and  stain  the  corpuscles  fora 
day  or  so  with  borax-carmine  and  mount  the  stained  corpuscles  in  glycerine  or 
glycerine-jelly. 

(h.)  Stain  another  specimen  with  very  dilute  eosin-hsematoxylin  (p.  70). 
The  haemoglobin  is  stained  by  the  eosin,  and  the  nuclei  by  the  haematoxylin. 
It  is  preserved  as  (a).  Other  combinations  of  dyes  will  suggest  themselves. 

(c.)  Make  similar  preparations  of  mammalian  blood. 

Farhenanaly,  Unters.  z.  Histologic  und  Klinik  d.  Blutes,  Berlin,  1891. 


III.] 


BLOOD-PLATELETS. 


123 


' © 

© 


Fig.  83.-  1,  2.  Coloured  corpuscles 
with  3.  Blood-plates ; 4.  Lymph 
corpuscle  surrounded  by  blood- 
plates. 


19.  Blood-Plates  or  Platelets  (H). — Wrap  a liandkercliief  round  a finger  to 
obtain  some  blood  (Lesson  111.,  p.  115).  On  the  skin  at  the  root  of  the  nail 
])lace  a drop  of  normal  saline  containing  methyl-violet  (.75  gram  in  1000  cc.). 
•Through  this  drop  prick  the  finger,  and  blood  runs  into  it.  Place  a little  of  the 
mixed  blood  and  methyl-violet  solution  on  a slide  ; cover  at  once  and  examine. 

It  requires  a good  microscope  and  carelul  observation  to  see  the  platelets. 
Tlie  red  corpuscles  are  unstained  while  the  colourless  corpuscles  are  stained.  In 
the  field  are  to  be  seen  small  oval,  refractive,  very  delicate,  non-nucleated 
bodies,  much  smaller  than  the  red  corpuscles 
— these  are  the  blood-plates  (fig.  83,  3).  They  ** 

are  about  2.5  in  diameter.  They  undergo 
changes  exceedingly  rapidly  in  shed  blood. 

Instead  of  methyl-violet  solution,  the  skin 
may  be  pricked  through  a drop  of  the  follow- 
ing mixture:  — i part  of  i per  cent,  osmic 
acid,  and  2 parts  .75  per  cent,  sodic  chloride. 

By  far  the  best  method  of  obtaining  blood- 
platelets  in  large  quantity,  and  in  a condition 
in  which  they  do  not  disappear,  is  to  allow 
blood  to  flow  into  a solution  of  oxalate  of 
potash  until  the  mixture  contains  at  least 
I per  1000  of  the  salt.  This  prevents  coagula- 
tion of  the  blood.  On  placing  such  blood  in  a 
centrifuge,  when  the  corpuscles  subside  a film  of  grayish  material  accumulates 
on  their  surface,  W’hich  consists  chiefly  of  blood-plates.  A drop  of  this  spread 
on  a slide,  dried,  and  stained  witli  a watery  solution  of  methyl-violet  5 B and 
mounted  in  balsam,  yields  permanent  prep.arations  of  these  bodies  {Moser). 

20.  Blood-Platelets — Dry  Method  (H). — Clean  a slide  thoroughly  ; sterilise 
it  in  the  flame  of  a Bunsen-burner,  and  allow  it  to  cool.  Obtain  a drop  of 
blood  from  the  finger  in  the  usual  way  ; get  a drop  on  the  slide,  and  with  the 
edge  of  another  slide  rapidly  spread  the  drop  of  blood  as  a thin  film  on  the 
sterilised  slide.  Move  the  slide  to  and  fro  in  the  air  until  the  film  of  blood 
dries.  The  whole  process  should  not  occupy  more  than  four  seconds.  Cover 
the  dry  film  with  a cover-glass  and  seal  the  latter  at  the  corners  with  paraffin. 

{a.)  Observe  the  coloured  blood-corpuscles,  which  for  the  most  part  retain 
their  shape,  and  between  them  the  blood-]datelets  or,  as  Hayem  calls  them, 
the  hsematoblasts.  They  are  readily  seen  thus  in  human  blood.  The  white 
corpuscles  are  somewhat  altered  in  shape. 

21.  Weigert’s  Method  of  Staining  Fibrin. — Embed  a thrombus  or  a ]>iece 
of  lung  affected  with  acute  croupous  pneumonia  in  celloidin,  and  make  a 
section.  Float  the  section  from  water  on  to  a slide.  Stain  it  for  ten  minutes 
or  so  with  a drop  of  the  following  fluid  : — 

Gentian  violet  (5  per  cent.)  . . 44  cc. 

Aniline  oil  ....  . i ,, 

Alcohol  (96  per  cent. ) . . . 6 , , 


Remove  the  stain  by  pressing  on  the  section  two  or  three  plies  of  blotting- 
paper,  or,  better,  unsized  printing-paper.  Add  a drop  of  iodine  in  iodide  of 
potassium  (iodide  of  potassium  5 per  cent,  and  saturated  with  iodine).  This 
quickly  decolorises  most  of  the  stained  parts.  Remove  the  iodine  with  blot- 
ting-paper in  the  same  way  as  before. 

Pour  on  the  section  a mixture  of  equal  volumes  of  xylol  and  aniline  oil, 
moving  the  mixture  over  the  preparation.  Remove  Ihis  and  apply  a fresh 
supply  of  the  xylol-anilim;  oil  mixture.  This  removes  all  the  water.  As 
long  as  there  is  a trace  of  whiteness  in  the  section  it  still  contains  water. 
After  all  the  water  is  removed,  dry  the  preparation  with  paper  as  before,  to 


124 


PRACTICAL  HISTOLOGY. 


[III. 


remove  as  much  aniline  oil  as  possible.  Wash  the  preparation  several  times 
with  xylol  to  remove  the  last  traces  of  the  aniline  oil  and  mount  in  xylol- 
balsam. 

The  stages  are  as  follows  : — 

1.  Harden  in  alcohol. 

2.  Stain  5-15  minutes  in  concentrated  aniline  water  solution  of  gentian 

violet. 

3.  Dry  with  blotting-paper. 

4.  Apply  iodine  solution  (2-3  mins.). 

5.  Dry  with  blotting-paper. 

6.  Decolorise  and  wash  in  aniline-xylol. 

7.  Remove  the  latter  and  mount  in  xylol-balsam. 

(a.)  Observe  the  threads  of  fibrin — very  fine  and  numerous — stained  a beau- 
tiful violet. 

22.  Solvent  Action  of  Serum.  — Place  some  blood  of  a rabbit  or  guinea-pig 
ill  a drop  of  blood  serum  of  a dog.  The  red  corpuscles  are  completely  dis- 
solved in  a few  minutes.  The  blood-corpuscles  of  a pigeon  or  frog  are  similarly 
but  more  slowly  dissolved,  except  the  nuclei.  This  property  of  dog’s  serum  is 
set  aside  by  previously  heating  the  serum  to  5o°-6o°  C.  for  about  half-an-hour. 


LESSON  lY. 

EPITHELIUM  (STRATIFIED)  AND  ENDOTHELIUM. 

Epithelium  presents  the  following  general  characters  : — 

1.  It  is  always  disposed  on  surfaces. 

2.  The  cells  are  united  by  cement. 

3.  There  are  no  blood-vessels  within  the  cells. 

Varieties  of  Epithelium. 

1.  Squamous. 

2.  Columnar. 

3.  Secretory. 

4.  Transitional. 

5.  Ciliated. 

Squamous  Epithelium  may  occur  either  in  a single  layer,  or  in 
several  layers;  in  the  former  case  it  is  sometimes  called  endothelium, 
in  the  latter  it  is  said  to  be  stratified. 

(A.)  In  a single  layer  it  lines  serous  and  synovial  membranes, 
heart,  blood-  and  lymph- vessels,  air-cells  of  lung,  pigmentary  layer 
of  retina,  posterior  surface  of  cornea,  anterior  surface  of  iri-;, 
membranes  of  brain  and  spinal  cord,  surfaces  of  tendons  and  tendon 
sheaths,  &c. 


IV.] 


EPITHELIUM  AND  ENDOTHELIUM. 


125 


As  endothelium^  it  consists  of  a single  layer  of  flattened  squamous 
transparent  cells  united  to  each  other  at  their  edges  by  means  of  a 
cement  substance.  • 

(B. ) As  stratified  squamous  epithelium,  it  covers  the  skin  (epi- 
dermis), and  lines  the  following  cavities  and  surfaces  : — mouth, 
pharynx  (lower  half),  oesophagus,  conjunctiva,  over  anterior  surface 
of  cornea,  vagina,  and  lower  half  of  cervix  uteri,  and  entrance  of 
urethra. 

I.  Isolated  Squamous  or  Scaly  Epithelium  Cells. — With  the 
finger-nail  or  a small  section-lifter  gently  scrape  the  inner  surface  of 
the  lip,  place  the  scrapings  on  a slide,  add  a drop  of  saliva, 
skimming  off  any  air-bubbles  with  a needle,  cover,  and  examine. 

1.  Squames  (H). — {a.)  Observe  large  flat  plates  or  squames  ^ 
floating  in  the  field,  either  singly  or  in  groups  ; the  cells  of  the  latter 
may  be  united  by  their  edges,  or  by  their  edges  and  surfaces. 
Select  a single  squame  seen  on  the  flat.  Note  its  large  size^  being 
five  to  ten  times  ])roader  tlian  a red  blood-corpuscle  ; its  polygonal 
shape ; the  colourless,  transparent,  and,  it  may  be,  slightly  granular 
body  of  the  cell,  and  the  small  oval  excentrically-placed  nucleus. 

(1).)  On  some  of  the  cells  fine  lines  may  be  seen,  some  of  them 
due  to  folds,  but  most  of  them  to  facets,  indicating  that  the  cell 
has  been  overlapped  and  slightly  indented 
by  its  neighbour.  Select  a group  of  cells 
where  the  cells  can  be  seen  adhering  to  each 
other  by  their  edges  and  surfaces,  as  the 
squames  occur  in  several  layers.  It  is  well 
also  to  look  for  a cell  seen  edgeways,  to 
observe  that  it  is  really  a flat  plate  (fig.  84). 

(e.)  Not  unfrequently  fungi,  such  as 
bacteria,  may  be  seen  adhering  to  the 
squames. 

{d.)  Salivary  Corpuscles  may  be  seen. 

They  are  sharply-defined  spherical  cells,  pro- 
vided with  a membrane,  and  about  the  size 
of  a colourless  blood-corpuscle.  They  contain 
fine  granules,  which  in  the  fresh  condition 
of  the  corpuscles  may  be  seen  to  exhibit 
Brownian  movement  (Lesson  I.  6).  Each  cell  may  contain  one 
small,  spherical,  excentrically-placed  nucleus,  or  sometimes  two 
nuclei  may  be  present.  They  seem  to  be  leucocytes  distended  with 
fluid. 

2.  Magenta. — Irrigate  the  preparation  with  a watery  solution  of 
magenta  (p.  74),  which  stains  deeply  the  nuclei  of  the  squames  and 
salivary  corpuscles,  while  the  peri-nuclear  parts  of  these  cells  are 
less  deeply  stained.  As  the  magenta  contains  alcohol,  the  latter 


Fig.  84.— Cells  of  Stratified 
Squamous  Epithelium  de- 
‘tached  from  the  Mouth. 
s.  Salivary  corpuscles. 


PRACTICAL  HISTOLOGY. 


126 


[IV. 


precipitates  the  mucin  of  the  saliva  either  in  the  form  of  fine  threads 
or  in  membranes,  which  are  stained  red  by  the  magenta. 

3.  Epidermis  of  Newt — Superficial  Layers  of  Stratified  Epithe- 
liimi  (L  and  H). — Keep  a newt  or  frog  for  a day  or  two  in  a small 
quantity  of  water,  and  do  not  change  the  water.  Very  soon  the  super- 
ficial layers  of  squames  will  be  “ cast as  thin  membranes.  Take 
these,  and  harden  them  in  absolute  alcohol.  Stain  a thin  piece  in 
hsematoxylin,  and  mount  it  in  balsam. 

(a.)  Try  to  get  a layer  sufficiently  thin,  so  that  the  cells  are  only 
one  layer  thick.  Note  the  polygonal,  large,  nucleated  cells  united  to 

each  other  by  their  edges  by  means 
of  a clear  cement  substance  (fig.  85). 

{b.)  The  nucleus  is  usually  ex- 
centric,  and  surrounded  by  slightly 
granular  material.  In  the  nucleus 
are  usually  two  or  three  nucleoli, 
and  sometimes  an  intra-nuclear 
plexus  of  fibrils  is  visible,  especially 
in  the  cells  of  the  deeper  layers. 
Sometimes  granules  of  the  pigment 
melanin  are  seen  in  the  cells,  especi- 
ally from  a dark-pigmented  newt. 

4.  V.S.  of  Stratified  Epithelium. 
Fig.  85.-Superficial  Layer  of  Squam^^^  — Xhis  may  be  conveniently  ex- 
Alcohol and  hajmatoxyiin,  x 300.  amined  either  in  a vertical  section 

of  the  skin  of  the  palm  of  the 
hand,  or  a similar  section  of  the  mucous  membrane  covering  the 
hard  palate  of  a cat  or  the  conjunctiva  on  the  cornea.  The  skin 
must  have  been  previously  prepared  by  being  hardened  in  absolute 
alcohol  or  chromic  acid  and  spirit  mixture  (p.  29),  while  the  mucous 
membrane  may  be  hardened  in  the  chromic  acid  and  spirit  or 
Muller’s  fluid. 

If  the  mucous  membrane  of  the  hard  palate  be  taken,  stain  it 
with  picro-carmine  and  mount  in  Farrant’s  solution.  Or  the  mucous 
membrane  may  be  stained  in  bulk  in  borax  carmine,  embedded  and 
cut  in  paraffin.  In  the  latter  case  the  sections  are  best  mounted  in 
balsam. 

(a.)  Examine  it  first  with  (L).  Observe  the  epithelium  arranged 
in  many  layers,  covering  a connective-tissue  basis — the  latter 
stained  red — and  projecting  in  the  form  of  fine  papillae  into  the 
epithelial  layer  (fig.  86).  Neglect  the  connective-tissue  basis 
meantime. 

(b.)  (i-i)  Select  a thin  part  of  the  deeper  layers  of  the  epithelium 
near  the  papillae,  and  note  that  the  cells  there  are  somewhat  small 
and  cylindrical,  with  their  nuclei  stained  red.  Study  the  change 


EPITHELIUM  AND  ENDOTHELIUM. 


IV.] 


127 


01 

loHsngsZ^ 


in  the  form  of  the  cells  towards  the  free  surface  of  the  epidermis 
(fig.  86),  where  they  become  corneous  and  less  granular. 

(c,)  Kote  in  the  deeper  layers  of  the  epithelium  prickle-cells. 
Adjacent  cells  are  connected  by  very  fine  processes  or  “ intercellular 
bridges/'  The  fine  spaces  between  the  bridges 
are  called  “ intercellular  channels.”  When 
these  are  broken  across  and  the  cells  isolated,  the 
cells  present  the  appearance  of  being  beset  by 
very  fine  processes,  and  hence  they  are  called 
“ prickle-cells.” 

(d.)  The  cells  vary  in  their  shape  and  characters 
from  below  upwards.  It  will  be  easy  to  detect 
the  ho7^y  layer  above,  composed  of  many  layers 
of  flattened,  hardened  cells.  This  forms  a fairly 
well-marked  layer,  the  cells  being  clearer  than 
those  situated  more  deeply;  the  nuclei  are  less 
conspicuous,  and  the  cells  generally  stain  yellowish 
with  picro-carmine. 

5.  Non-Corneous  Stratified  Epithelium  from 
the  (Esophagus  (H). — Macerate  the  mucous 
membrane  of  the  oesophagus  of  a calf  or  other 
animal  for  a week  in  per  cent,  bichromate  of 
potash.  This  “dissociates”  the  epithelial  cells, 
so  that  when  the  surface  is  scraped,  one  obtains 
isolated  cells.  Make  a cover-glass  preparation 
(p.  114),  and  stain  it  in  aniline- water-gentian- 
violet,  f.6.,  gentian-violet  solution  dropped  into 
aniline  water  (p.  73).  Dry  and  mount  in  balsam. 

{a.)  Observe  the  isolated  squamous  cells,  each  with  a small  ex- 
centrically-placed  nucleus  stained  of  a violet  tint.  Numerous  facets 
are  seen  in  the  cells,  and  their  shape  both  on  the  flat  and  on  edge 
can  be  carefully  studied. 

(h.)  If  desired,  stain  some  in  picro-carmine.  This  is  best  done  by 
keeping  them  in  the  picro-carmine  on  a slide  placed  under  a bell-jar, 
with  water  to  prevent  evaporation,  in  a moist  chamber. 

6.  Horny  Epidermis. — Macerate  a shred  of  epidermis  in  35  per 
cent,  caustic  potash.  After  a time  it  is  softened  and  can  be  broken 
up  with  needles.  The  cells  fall  asunder  and  swell  up  in  the  fluid, 
and  appear  as  spheroidal  cells  with  a membrane,  but  no  nucleus  is 
visible.  Examine  them  in  the  potash  solution.  No  water  must  be 
added,  else  the  cells  are  dissolved. 

7.  Prickle  Cells  (H). — Place  for  twenty-four  hours  in  i per 
cent,  osmic  acid  a small  piece  of  the  palmar  surface  of  the  skin 
— less  than  one-eighth  of  an  inch  cube — from  a freshly-amputated 
finger.  Make  vertical  sections  by  freezing,  or  after  embedding  in 


FIG.  86.— V.S.  Mucous 
Membrane  of  Hard 
Palate  of  Cat.  Epi- 
dermis with  cor- 
neous and  deeper 
layers  with  prickle 
cells.  Below,  con- 
nective tissue  of 
the  mucous  mem- 
brane with  a papilla. 
Chromic  acid  and 
spirit,  picro  - car- 
mine, Farrant’s 
solution. 


PRACTICAL  HISTOLOGY. 


1 28' 


[IV. 


paraffin,  and  mount  the  former  in  Warrant’s  solution  and  the  latter 
in  balsam. 

{a.)  Observe  the  2^i*i<^ble-cells  in  situ,  i.e.,  polygonal  cells  in  the 
deeper  layers  of  the  rote  mucosum,  with  fine  processes  connecting 
adjoining  cells,  leaving  thus  a system  of  fine  spaces  between dhe  cells 
(fig.  87).  The  fine  fibres  which  pass  from  cell  to  cell  form  ‘‘inter- 
cellular bridges/^  and  when  these  bridges  are  broken  across  they 
give  the  appearance  as  if  the  cells  were  beset  with  fine  prickles. 

8.  Isolated  Cells  from  the  Different  Layers  of  the  Epidermis 
(H). — It  is  usual  to  macerate  very  small  pieces  of  any  membrane 
covered  with  stratified  epithelium  in  -J-  or  per  cent,  of  potassic 
bichromate,  which  usually  takes  more  than  a week  to  dissociate  the 
cells. 


Fig.  87. — Prickle-Cells  from  the 
Deeper  Layers  of  the  Epi- 
dermis of  the  Palm,  showing 
intercellular  bridges  and 
channels.  Osmic  acid. 

A much  speedier  method  is  that  of  Schiefferdecker.  Make  a 
watery  extract  of  “ pancreaticum  siccum  of  Dr.  Witte  of  Rostock. 
Filter,  and  in  the  filtrate  place  a small  fragment  of  fresh  skin  or 
the  pad  from  the  upper  jaw  of  a sheep.  Place  the  fluid  near  the 
fire  or  in  an  oven  at  40°  C.  Within  four  hours,  the  epidermis  can 
be  detached,  and  its  cells  fall  readily  apart.  Preserve  it  in  a test- 
tube  in  a mixture  of  equal  parts  of  water,  alcohol,  and  glycerine. 
It  forms  a deposit  at  the  bottom  of  the  tube.  A little  of  the  deposit 
is  mounted  in  glycerine  or  Farrant’s  solution,  covered  and  examined. 
It  may  be  stained  with  picro-carmine  or  methylene-blue. 

(a.)  Observe  numerous  cells  of  different  shapes,  some  flattened, 
otliers  cubical,  and  many  “prickle-cells^^  (fig.  88).  Many  of  the 
cells  exhibit  facets  where  they  have  been  pressed  against  each 
other. 

9.  Isolated  Prickles  and  other  Cells  from  the  Pad  of  a Sheep’s 
Mouth  (H)  . — Isolated  by  means  of  “ pancreaticum  siccum”  (y. 
supra).  Many  of  the  somewhat  cubical-shaped  cells  show  the 
“ prickles  ” beautifully.  These  cells  may  be  stained  with  an  aniline 
dye  or  picro-carmine,  and  some  of  them  show  two  nuclei. 


Fig.  88. — Prickle  Cells  Isolated  from 
the  Human  Epidermis  by  means  of 
Iodised  Serum.  n.  Prickles ; d. 
Space  between  nucleus  and  cell- 
body,  X 800. 


1 


IV.] 


EPITHELIUM  AND  ENDOTHELIUM. 


129 


Tig.  89. — Endothelium  of  the 
Peritoneal  Surface  of  the 
Central  Tendon  of  the 
Diaphragm  of  a Rabbit. 
Silvered.  /.  Lymphatic 
slit;  t.  Tendon;  c.  Ordi- 
nary endothelial  cells ; n. 
Islands  of  small  cells. 


10.  Endothelium  of  Central  Tendon  of  Diaphragm. — Mount 
ill  balsam  a small  piece  of  the  central  tendon  of  the  diaphragm  of 
a rabbit  stained  in  silver  nitrate  (Method, 

P-  77)- 

- {a.)  Observe  the  polygonal  areas  which 
map  out  the  outlines  of  the  single  layer  of 
squames  covering  the  surface  of  the  tendon. 

These  areas  are  bounded  by  brown  or  black 
lines,  the  so-called  ‘‘silver  lines’"  (fig.  89). 

As  a rule,  no  nucleus  is  visible  in  the  cells, 
but  it  may  be  brought  into  view  by  staining 
with  logwood  and  mounting  the  preparation 
in  Canada  balsam. 

(J).)  Amongst  these  may  be  seen  small 
groups  or  islands  of  smaller  granular  cells. 

In  some  specimens  stomata  surrounded  by 
granular  or  germinal  cells  may  be  seren. 

These  stomata  open  into  a plexus  of  lym- 
phatic capillaries  in  the  substance  of  the 
tendon  (Lesson  on  Lymphatics). 

11.  Omentum  of  Young  Eabbit  (L  and 
H). — Mount  in  balsam  a small  piece  of  the 
silvered  omentum  of  a young  rabbit  (one  week  old)  (Method,  p.  77). 

(a.)  Observe  a thin  fibrous  membrane  mapped  into  polygonal 
areas — some  of  them 
with  more  or  less 
sinuous  outlines — by 
means  of  black  “ silver 
lines (fig.  90). 

The  omental  mem- 
brane composed  of 
connective  tissue  is 
covered  on  both  sur- 
faces with  a continu- 
ous layer  of  endo- 
thelium. 

{h.)  Kaise  the  lens 
by  means  of  the  fine 
adjustment  until  the 
upper  layer  of  squames 
comes  distinctly  into 
view;  then  depress  the 
lens  and  focus  through 
the  thickness  of  the  membrane  to  bring  into  view  the  layer  of 
squames  covering  the  deeper  surface,  and  note  that  the  outlines  of 
13  I 


Fig.  90. — Omentum  of  a Young  Rabbit  stained  v^^ith  Silver 
Nitrate  (x  300),  showing  the  epithelium  on  the  upper 
and  under  surfaces  ; the  outlines  of  the  latter  faintly 
indicated.  Some  holes  are  also  seen  in  it. 


130 


PRACTICAL  HISTOLOGY. 


[IV. 


the  cells  do  not  correspond  with  those  of  the  upper  layer.  In 
focussing  through  the  membrane,  note  the  plexus  of  • elastic  fibres 
in  it. 

(c.)  To  see  the  nuclei  of  tne  endothelial  cells,  stain  another  piece 
of  the  silvered  omentum  in  logwood  and  mount  it  in  balsam. 

The  omentum  of  a young  rabbit  is  chosen  because  it  is  nearly  a 
complete  membrane  with  few  fenestrse  or  holes  in  it. 

12.  Omentum  of  Cat  (l  and  H). — Mount  in  balsam  a small 
piece  of  the  silvered  omentum  of  a cat  (Method,  p.  77).  In  cutting 
the  omentum  into  small  pieces,  the  easiest  way  is  to  spread  it  out,  or 
rather  float  it  out,  on  a sheet  of  paper,  and  then  cut  the  paper  and 
omentum  into  pieces  of  the  necessary  size.  The  pieces  are  thus 

less  liable  to  fold  up,  and  are 
more  readily  manipulated  on 
the  slide. 

(a.)  (L)  Observe  a mesh 
work  of  trabeculae  (T)  bound- 
ing open  polygonal  spaces  (rig. 
91,  m).  In  the  larger  tra- 
beculae may  be  seen  blood- 
vessels (c)^  an  artery  or  a 
vein,  or  both,  surrounded 
here  and  there  by  groups  of 
large  clear  cells — fat-cells  (/). 
All  the  trabeculae  are  com- 
pletely covered  with  endo- 
thelial cells,  whose  outlines, 
mapped  out  by  silver  lines, 
can  just  be  recognised. 

(b.)  (H)  Select  a large 
strand  with  a blood-vessel  in 
corpuscles.  Silver  nitrate  and  hsematoxylin,  (T)  Focus  the  silver  lines 
X 100.  / \ • 

(s)  on  its  upper  surface,  and 
gradually  depress  the  lens  until  the  fibrous  tissue  composing  the 
strand  comes  into  view,  and,  still  lowering  the  lens,  bring  the 
endothelium  on  the  under  surface  into  view. 

(c.)  Select  a fine  trabecula,  and  note  the  silver  lines  on  it;  also 
observe  the  fibrous  tissue  of  which  it  is  composed. 

(d.)  If  desired,  a preparation  may  be  stained  with  logwood  to 
reveal  the  nuclei  of  the  cells  of  the  endothelium  (a),  as  well  as 
those  of  the  fibrous  tissue  composing  the  membrane  (b).  The 
nuclei  of  the  endothelial  cells  are  superficial,  and  usually  spherical 
(a)  ; those  of  the  connective-tissue  corpuscles  are  in  the  substance 
of  the  membrane,  and  are  usually  more  flattened  and  somewhat 
oval  (b). 


Fig.  91. — Omentum  of  Cat  Silvered,  t = Trabe- 
cula, with  c.  Blood-vessel ; /.  Fat-cells ; s. 
Silver  lines,  and  a.  Nuclei  of  the  endothelium; 
m.  Meshes ; b.  Nuclei  of  the  connective-tissue 


EPITHELIUM  AND  ENDOTHELIUM. 


^r.] 


131 


ADDITIONAL  EXERCISES. 

13.  Dogiel’s  Methylene-Blue  Methd^HRace  any  fresh  membrane,  e.g.^ 
omentum,  mesentery,  capsule  of  kidue}'^,  m^4  per  cent,  solution  of  methylene- 
blue  in  normal  saline.  Allow  it  to  stain  for  ten  minutes  or  so.  Wash  ittwic;e 
in  a saturated  watery  solution  of  picrate  of  ammonia  and  examine  in  glycerine. 
The  outlines  of  the  cells  are  stained  of  a purplish  colour.  This  method  may 
be  used  instead  of  the  silver  method  to  demonstrate  the  existence  of  endo- 
thelium on  any  surface,  e.g.,  on  tendons  (rat)  in  blood-  or  lymph-vessels.  It 
is  also  used  to  demonstrate  lymph-spaces. 

14.  Sections  covered  by  or  consisting  of  epithelial  cell  structures  may 
be  stained  in  a weak  watery  solution  of  benzo-azurin.  This  stain  is  not 
removed  by  alcohol,  and  the  sections  can  be  mounted  in  balsam.  Or  benzo- 
purpurin  (B. ) may  be  used.  This  gives  a reddish  stain,  any  excess  being 
removed  by  alcohol  rendered  feebly  alkaline,  e.g.^  by  lithium  carbonate. 
Benzo-azurin  stains  connective  tissue  of  a bright  blue. 


LESSON  y. 

COLUMNAR,  SECRETORY,  AND  TRANSITIONAL 
EPITHELIUM. 

Columnar  Epithelium  lines  the  mucous  membrane  of  the  ali- 
mentary canal  from  the  cardiac  orifice  of  the  stomach  onwards ; 
the  greater  part  of  the  ducts  of  the  glands  opening  into  it ; other 
gland  ducts. 

II.  Columnar  Epithelium. — Slit  open  the  small  intestine  of  a 
rabbit  or  a kitted.  Wash  the  mucous  surface  with  normal 

saline  to  remove  any  adherent  particles. 

1.  Fresh  Condition  (H). — With  a scalpel  gently  scrape  the 
mucous  surface  and  transfer  what  is  on  the  scalpel  to  a drop  of 
normal  saline  solution  on  a slide.  Diffuse  the  scrapings  in  this 
fluid,  and  to  prevent  the  pressure  of  the  cover-glass,  place  in  the 
fluid  a hair  half-an-inch  in  length.  This  preparation  is  not  to  be 
preserved. 

{a.)  Observe  numerous  columnar  epithelial  cells,  a few  isolated, 
but  most  of  them  adhering  together.  Side  View  of  the , Cells.- — 
Select  an  isolated  cell,  notice  its  columnar  form,  usually  tapering 
to  a blunt  point  at  one  end,  while  the  body  of  the  cell  is  faintly 
granular,  and  contains  a clear  oval  nucleus.  The  free  broad  end 
is  covered  with  a highly  retractile  disc — seen  on  edge  as  a narrow 
retractile  band — with  fine  vertical  striae  in  it  (fig.  92). 


132 


PRACTICAL  HISTOLOGY 


[V- 


Many  more  or  less  complete  villi  may  be  seen.  Along  the  edge 
of  the  villus,  covered  by  its  layer  of  columnar  cells,  the  clear, disc 
is  readily  seen,  and  by  focu^^BfcUie  surface  of  a villus  the  nucleated 
mosaic  formed  by  the  ends  oHRe  cells,  with  the  rounded  mouths 
of  the  goblet-cells,  come  into  view. 

(b.)  If  the  animal  was  killed  whilst  its  food  was  undergoing 
digestion,  refractive  fatty  granules  may  be  seen  in  the  protoplasm 
of  the  cells. 

(^c.)  End  View  of  the  Cells. — Move  the  preparation  until  a 
fragment  of  detached  epithelium  is  seen  showing  the  free  ends  of 
the  cells  directed  towards  the  observer 


(fig,  92,  d). 

(d.)  Note  in  such  a group  of  cells  the 
polygonal  outlines  of  the  ends  of  the 
cells,  and  in  each  polygonal  area  a large 
spherical  nucleus,  which  appears  almost 


Fig.  92.— g,  &.  Isolated  Columnar  to  fill  the  area.  A nucleolus 


Epithelial  Cells  from  the  Small 
Intestine  of  Cat ; c.  Goblet-cell ; 
d.  Ends  of  columnar  cells  and 
open  mouths  of  goblet -cells 
directed  towards  observer. 


often 


be  seen.  Here  and  there  may  be  seen 
the  rounded  opening  of  a goblet-cell. 
Focus  carefully  and  notice  the  appear- 
ance of  the  goblet-cell.  When  the  open 
mouth  is  in  focus,  it  is  seen  as  a circle  of  small  diameter,  and  on 
depressing  the  tube  the  broad  part  of  the  cell  comes  into  view  (fig. 
92,  d). 

(e.)  Amongst  the  cells  may  be  found  isolated  goblet-cells  (fig. 
92,  c).  Each  cell  has  an  open  mouth,  while  the  lower  part  of  the 
cell  contains  a nucleus  embedded  in  a small  quantity  of  protoplasm. 

2.  Isolated  Columnar  Epithelium  of  Newt  (H). — Mount  in 
glycerine  a small  quantity  of  isolated  columnar  cells  which  have 
been  dissociated  by  maceration  in  dilute  alcohol  and  subsequently 
stained  with  picro-carmine.  Place  a short  length  of  hair  under  the 
cover-glass. 


The  small  intestine  of  a rabbit  may  be  used,  but  far  larger  cells 
are  obtained  from  the  intestine  of  a newt.  Macerate  the  whole 
intestine  in  dilute  alcohol  for  twenty-four  hours  and  stain  in  bulk 
in  picro-carmine  for  at  least  another  twenty-four  hours.  On 
scraping  the  mucous  surface,  the  cells  are  detached  and  diffused  in 
formic  glycerine. 

(a.)  Observe  the  large,  tall,  columnar  cells,  often  tapering  to  a 
point  at  their  lower  end,  the  red-stained  nucleus,  and  the  clear 
striated  disc.  The  nucleus  usually  exhibits  a distinct  nucleolus 
(fig.  93),  and  sometimes  two  nuclei  are  present.  Sometimes  two 
nucleoli  are  seen  in  a nucleus,  and  the  cell  itself  may  be  branched 
at  its  fixed  extremity. 

If  the  intestine  of  a newt  be  macerated  for  twenty-four  hours  in 


V.]  COLUMNAR  EPITHELIUM.  1 33 

5 per  cent,  ammonium  chromate,  then  the  nuclei  and  cell-body  show 
a distinct  fibrillar  structure.  _ 

III.  Glandular  Epithelium  oc^fl^  the  secretory  glands,  e.g.^ 
liver,  pancreas,  salivary,  gastric,  JntSIKl,  and  other  glands.  Keces- 
sarily  it  must  vary  very  greatly  in  sliape  and  in  its  functions. 

3.  Secretory  or  Glandular  Epithelium  of  Liver. — With  a 
clean  scalpel  scrape  the  cut  surface  of  the  liver  of  an  animal  just 
killed,  e.g.,  a rat.  Place  the  scrapings  in  dilute  alcohol  (twenty- 
four  hours),  pour  off  the  alcohol  and^cover  it  with  picro-carmine 
(twenty-four  hours). 

Liver-Cells  (H). — (a,)  Examine  the  isolated  cells  in  glycerine 
with  the  usual  precautions.  Observe  the  cubical  cells,  which  may 
be  isolated  or  adhering  in  groups  of  two  or  three.  The  granular 


Fig.  93. — Isolated  Columnar 
Epithelial  Cells  from  the 
Newt’s  Intestine.  Dilute 
alcohol  and  picro-car- 
mine, X 300. 


Fig.  94.— Isolated  Hepatic  Cells,  d.  With  two 
nuclei ; h.  Oil-drops  ; c.  Isolated  nucleus  of  a 
cell.  Teased  fresh. 


protoplasm  is  stained  yellowish,  and  each  cell  has  a spherical  bright- 
red  nucleus.  The  protoplasm  may  contain  globules  of  oil,  and 
occasionally  two  nuclei  may  be  seen  in  a cell,  especially  in  the  liver- 
cells  of  a young  rat  (fig.  94). 

{h.)  If  the  cells  be  much  broken  up,  liberated  nuclei  and  granules 
of  oil,  and  sometimes  red  blood-corpuscles,  are  seen  in  the  field. 

(c.)  Acetic  acid  clears  up  the  protoplasm,  makes  the  nucleus  more 
distinct ; it  does  not  affect  any  fatty  particles,  but  merely  makes 
them  more  evident. 

Sulpho-cyanide  of  potassium  (5  per  cent.)  is  an  excellent  medium 
for  maceration  of  the  liver-cells.  The  plexus  of  fibrils  in  the  nuclei 
is  thus  rendered  visible. 

IV.  Transitional  Epithelium  occurs  in  the  urethra,  urinary 
bladder,  ureters,  and  pelvis  of  the  kidney.  It  is  confined  to  the 
gen ito-ur inary  mucous  membrane.  It  consists  of  several  (2-3-4) 
layers  of  cells.  The  superficial  cells  are  large  and  flattened 
(especially  if  the  bladder  has  been  kept  distended),  often  with  two 
nuclei,  and  with  depressions  on  their  under  surface  produced  by  the 
large  rounded  ends  of  the  cells  of  the  next  layer.  The  cells  of  the 


PRACTICAL  HISTOLOGY. 


134 


[V. 


next  layer  are  somewhat  pyriform  (fig.  95,  &),  while  the  deepest  layers 
are  composed  of  smaller  polylmdral  cells. 

4.  Transitional  Epithel^^^bf  Bladder  (H). — Place  the  dis- 
tended bladder  of  a frog  or'^c^  in  dilute  alcohol  for  twenty-four 
hours,  stain  it  en  masse  for  the  same  time  in 
picro-carmine,  scrape  off  a little  of  the  mucous 
surface  and  diffuse  it  in  glycerine,  add  a hair 
and  cover.  The  cells  may  also  be  macerated 
by  using  instead  ^ per  cent,  bichromate  of 
potash  solution. 

(a.)  Observe  various  forms  of  cells,  some 
of  them  more  or  less  flattened  with  facets  on 
their  surfaces  (fig.  95,  a,  a'),  others  elongated 
with  finger-shaped  processes  (&),  some  pear- 
shaped,  and  others  cubical.  All  are 
nucleated. 

5.  To  Distend  a Frog’s  Bladder. — By 

means  of  a pin  transfix  the  skin  at  the 
margins  of  the  anus,  and  tie  round  the  pin 
a thread  so  as  to  completely  occlude  the 
aperture.  Open  the  abdomen,  make  a slit 
into  the  rectum,  and  from  the  latter,  after  removing  its  contents, 
inject  dilute  alcohol  (p.  25)  into  the  bladder.  When  the  bladder  is 
distended,  ligature  it,  cut  it  out,  and  suspend  it  in  its  inflated  con- 
dition in  dilute  alcohol  for  twenty-four  hours. 

If  the  bladder  of  a cat  or  guinea-pig  be  used,  it  is  distended  from 
the  urethra  with  dilute  alcohol,  and  suspended  for  twenty-four 
hours  in  a large  volume  of  the  same  liquid. 


Fig.  95.  — Isolated  Transi- 
tional Cells  from  the 
Bladder  of  a Guinea-pig. 
a.  A superficial  cell  seen 
from  the  side,  and  a'  from 
below;  fe^nd  c.  Cells  from 
the  deeper  layers.  Dilute 
alcohol  and  picro-car- 
mine,  x 300. 


ADDITIONAL  EXEKCISES. 

6.  To  Silver  the  Free  Ends  of  Columnar  Epithelium. — A small  piece  of 
the  mucous  surface  of  the  small  intestine  of  a cat  is  washed  in  distilled  water, 
and  then  placed  for  ten  minutes  in  a J per  cent,  silver  nitrate  solution,  and 
silvered  in  the  usual  way  (Method,  p.  77).  After  hardening  in  alcohol,  if  tiie 
epithelium  be  detached  and  mounted  in  glycerine,  it  is  easy  to  obtain  a view 
of  the  free  ends  of  the  epithelial  cells,  with  the  cement  substance  between  them 
indicated  l)y  “silver  lines,”  and  also  to  see  the  open  mouths  of  the  goblet-cells. 
The  view  obtained  is  that  shown  in  fig.  92,  d, 

N.B. — Other  preparations  of  these  forms  of  epithelium  will  be  obtained  in 
sections  of  the  organs  in  which  they  occur.  • 


CILIATED  EPITHELIUM. 


135 


vl] 


LESSON  VI. 

CILIATED  EPITHELIUM. 

Ciliated  Epithelium  occurs  in  the  nasal  mucous  membrane 
(except  that  of  the  olfactory  region),  the  cavities  accessory  to  the 
nose,  the  upper  half  of  the  pharynx,  the  Eustachian  tube,  larynx, 
trachea,  and  bronchi,  the  uterus  (except  the  lower  half  of  the  cervix), 
Fallopian  tubes,  vasa  efferentia  to  lower  end  of  epididymis,  the 
ventricles  of  the  brain,  and  the  central  canal  of  the  spinal  cord. 

V.  Ciliated  Epithelium. — A ciliated  cell  may  be  any  shape,  but 
usually  it  is  more  or  less  columnar.  Only  the  cells  of  the  super- 
ficial layer  hear  cilia.  The  bunch  of  cilia  are  directly  continuous 
with  the  protoplasm  of  the  interior  of  the  cell,  and  are  planted  on  a 
clear  disc,  which  is  said  to  be  composed  of  small  “ knobs  })lacedside 
by  side  to  form  a bright  retractile  disc  ; a cilium  being  attached  to 
each  knob.  The  deeper  cells  may  be  pyriform  or  polyhedral,  accord- 
ing to  the  situation  from  which  the  epithelium  is  obtained.  Goblet- 
cells  may  be  found  between  the  superficial  ciliated  cells. 

1.  Ciliary  Motion  in  the  Frog  (H). — After  pithing  a frog,  gently 
scrape  the  roof  of  its  mouth  with  a scalpel,  and  diffuse  the  scraping 
in  a drop  of  normal  saline,  add  a short  piece  of  hair,  and  cover. 

(a.)  Notice  groups  of  cells  with  cilia  on  their  free  surface.  The 
cilia  bend  quickly,  at  the  rate  of  ten  to  twelve  times  per  second,  with 
a whip-like  motion  in  one  direction,  and  then  rapidly  unbend,  thus 
creating  currents  in  the  liquid,  and  thereby  moving  the  corpuscles, 
granules,  or  other  free  particles  that  may  be  present.  They  bend 
more  rapidly  in  one  direction  than  the  other.  All  the  cilia  covering 
one  surface  are  not  in  motion  at  one  time,  but  the  movement  passes 
from  cell  to  cell  in  a wave-like  form.  If  a portion  of  cell  with  cilia 
attached  is  in  view,  the  fragment  of  the  cell  may  be  seen  to  be 
moved  in  a definite  direction  by  the  vibratile  motion  of  its  own 
cilia.  Cilia  detached  from  a cell  cease  to  move. 

2.  Ciliary  Motion  in  the  Mussel  (L  and  H). — Open  a salt-water 
mussel,  collect  the  salt  water  which  escapes,  cut  out  a fragment  of 
one  of  the  flattened  yellow  gills,  and  place  it  in  a drop  of  salt  water. 
By  means  of  two  needles  separate  slightly  the  bars  wliich  compose 
the  gills,  cover  and  examine. 

(a.)  (L)  Observe  the  bars  with  their  free  rounded  ends  and  their 
surfaces  beset  with  a fringe  of  moving  cilia,  which  cause  the  ])articles 
suspended  in  the  fluid  to  be  carried  along  in  a definite  direction. 


136 


PRACTICAL  HISTOLOGY. 


[VI. 


(h.)  (H)  Select  a single,  cilium  observe  that  it  is  a clear  homo- 
geneous tapering  filament,  placed  on  a clear  band  which  covers  the 
cells  on  the  surface  of  the  bar  of  the  gill.  Notice  how  the  cilium 
bends  more  at  the  top  than  base,  and  how  it  straightens  itself  again. 
The  movement  may  go  on  for  several  hours.  The  backward  move- 
ment is  less  rapid  than  the  forward  stroke. 

3.  Heat  (H). — By  means  of  a camel-hair  brush  run  a ring  of  oil 


Fig.  96. — Slide  with  a Ring  of  Glass  Tube  fixed  to  it,  for  Studying  the  Action  of 
Chloroform  on  Cilia. 

round  the  preparation  (2),  and  put  it  aside  for  an  hour  or  so,  until 
the  ciliary  motion  becomes  slower. 

(a.)  Place  the  slide  on  a hot  stage  (fig.  75)  and  gradually  apply 
heat.  As  the  cilia  are  warmed  they  move  more  quickly;  but  if  the 
temperature  be  too  high,  of  course  the  proteids  are  coagulated  and 
the  cells  killed.  If,  while  the  cilia  are  moving  rapidly,  the  source 
of  heat  be  removed,  as  the  preparation  cools  the  cilia  gradually  move 
more  and  more  slowly. 

Use  the  hot  stage  described  in  Lesson  III.  5.  But  in  using  it, 
put  the  preparation  of  cilia  on  a cover-glass  moistened  with  a drop 
of  sea-water,  and  invert  the  cover-glass  over  the  aperture  in  the  hot 
stage,  so  that  the  drop  of  fluid  and  cilia  hang  in  the  little  circular 
cavity. 

4.  Weak  Alkalies. — To  a preparation  in  which  the  cilia  move 
languidly,  apply  a drop  of  \ per  cent,  solution  of  caustic  potash. 
This  immediately  revives  their  action  for  a short  time ; but  as  the 
alkali  penetrates  into  the  cells,  it  ultimately  kills  them. 

5.  Chloroform  (L  and  H). — Place  a fragment  of  a mussehs  gill  in 
a drop  of  salt  water  on  a cover-glass.  Put  a small  drop  of  chloroform 
in  a glass  cell  and  place  the  cover-glass  on  the  cell,  with  the  drop  of 
fluid  hanging  into  the  latter,  as  shown  in  fig.  96. 

(a.)  (H)  Observe  the  movement  of  the  cilia,  and,  as  the  chloroform 
vapour  diffuses  into  the  drop  of  water  and  acts  on  the  cilia,  how 
they  move  slower  and  slower.  If  the  action  of  the  chloroform  be 
pushed  too  far,  their  movement  will  be  arrested.  If  the  action  of 
the  chloroform  be  not  too  prolonged,  and  the  preparation  removed 
and  freely  exposed  to  the  air,  the  cilia  may  begin  to  move  again. 

6.  Action  of  Gases  on  Ciliary  Motion,  e.y..  Carbon  Dioxide. — 

Carbonic  acid  is  generated  in  the  usual  way  in  a flask  containing 


CILIATED  EPITHELIUM. 


137 


VI.] 


marble  and  dilute  hydrochloric  acid,  and  by  means  of  a caoutchouc 
tube  it  is  conducted  to  a glass  gas-chamber  (fig.  97,  C),  over  whmh 
the  preparation  of  cilia  on  a cover-glass  is  inverted. 

If  it  be  preferred,  the  following  moist  chamber,  by  Ranvier,  for 
studying  the  action  of  gases  may  be  used.  It  consists  of  a brass  box 


Fig.  97.— Gas-Chamber  for  Studying  the  Action  of  Gases  on  Cilia.  A.  Inlet ; 
B.  Outlet-tube;  C.  Glass  gas-chamber. 


about  the  size  of  a microscopical  slide,  and  perforated  at  the  centre 
by  an  aperture  2 cm,  wide,  which  is  closed  below  by  a plate  of  glass. 


Fig.  98.~Ilanvier’s  !Moist  Chamber  for  Api)lying  Gases  to  a [’reparation. 


i 


In  the  centre  of  the  aperture  is  fixed  a plate  of  glass  (fig.  98,  a),  less 
in  diameter,  thus  leaving  a circular  trench  all  round  (b).  Moreover, 
the  height  of  this  circular  plate  of  glass  is 
less  than  the  height  of  the  brass  box  by  at 
least  xV^h  mm.  The  box  is  perforated  by 
two  tubes,  through  which  the  gases  can 
be  conducted  to  the  ]3reparation,  which  is 
placed  between  the  top  of  the  circular 

glass  disc  and  the  cover-glass  which  covers  ^ 

9 1 , 1 .1  . • 1 Pig.  99.  Canons  Forms  of 

111  completely  the  aj^erture  in  the  brass  ciliated  Cells  from  the 

box.  In  this  way  the  gas  or  vapour  can  H^"paia{e“LpLgus 

be  applied  to  a preparation  still  in  a normal  of  the  Frog.  Dilute  alcohol 
fluid  medium.  x 3- 


(a.)  If  carbon  dioxide  be  used,  observe  that  it  rapidly  arrests  the 
movement  of  the  cilia  and  renders  the  cells  granular,  probably  from 
the  precipitation  in  them  of  paraglobulin. 


138 


PRACTICAL  HISTOLOGY. 


[VI. 


7.  Isolated  Ciliated  Epithelium  and  Goblet-Cells  (Frog)  (H). — 

Scrape  off  a little  of  the  epithelium  from  the  mucous  membrane  of 
the  palate  of  a frog,  which  has  been  macerated 
for  twenty-four  hours  in  dilute  alcohol  and  after- 
wards stained  by  picro-carmine.  Before  staining, 
it  is  advisable  to  place  the  isolated  cells  for 
several  hours  in  | per  cent,  osmic  acid.  Diffuse 
the  cells  in  glycerine,  put  in  a hair,  cover,  and 
examine. 

(a.)  Observe  an  isolated  ciliated  cell ; it  is 
short  and  columnar,  perhaps  tapering  or  divided 
at  one  end,  while  the  other  end  is  beset  with 
cilia,  resting  on  a clear,  transparent,  refractile 
disc ; the  protoplasm  is  granular,  and  encloses  an 
oval  red-stained  nucleus  with  one  or  two  bright 
excentrically-placed  nucleoli  (fig.  99). 

Besides  the  ciliated  cells,  there  are  others 
without  cilia,  oval  or  elongated,  pointed  at  one 
end.  These  are  from  the  deeper  layers  of  the 
ciliated  surface  (fig.  102). 

(b.)  Numerous  goblet  or  chalice  cells  will  also 
be  seen.  They  are  cup-shaped  cells,  with  an  open 
mouth,  and  containing  mucigen.  There  is  a 
small  amount  of  protoplasm  at  one  end  of  the  cell 
— the  end  by  which  it  is  fixed — which  encloses 
a spherical,  oval,  or  compressed  nucleus.  The 
goblet-cells  may  be  seen  in  two  conditions,  some  clear  with  their 
mucigen  discharged,  and  others  full  of  granules  or 
‘‘  loaded  with  mucigen. 

The  cells  may  also  be  isolated  or  dissociated  by 
macerating  the  membrane  in  iodised  serum  (p.  25). 
A cell  isolated  in  this  way,  and  magnified  1000 
diameters,  is  shown  in  fig.  100. 

8.  Isolated  Ciliated  Cells  (Mammal). — Use  the 
trachea  of  a cat  or  other  mammal,  and  macerate 
small  pieces  in  dilute  alcohol  (twenty-four  hours). 
Stain  it  in  bulk  in  picro-carmine.  The  isolated  cells 
are  examined  in  glycerine  or  glycerine-jelly.  If  the 
trachea  of  the  ox  be  used,  observe 

(a.)  The  tall  narrow  ciliated  cells  (fig.  loi),  each 
with  its  cilia  and  clear  disc.  The  ends  of  the  cells 
may  be  pointed  or  branched.  Amongst  these  may 
be  seen  oval  or  battledore-shaped  cells  (a),  the 
younger  cells  which  exist  in  the  deeper  layers  of  the  mucous 
membrane. 


Fig.  ioo.  — Isolated 
Ciliated  Cell  from  the 
OEsophagus  of  a Frog. 
c.  Cilia ; p.  Clear 
disc ; n.  Nucleus  ; 
m.  Irregular  extre- 
mity. Iodised  serum, 
X 1000. 


Fig. 


. — Ciliated 
Cells  from  Tra- 
chea of  Ox.  a. 
Cell  from  the 
deeper  layers. 
Dilute  alcohol 
and  picro-car- 
mine, X 300. 


VI.] 


CILIATED  EPITHELIUM. 


139 


9.  Ciliated  Epithelium  (L  and  H ). — Mount  a vertical  section  of 
the  respiratory  mucous  membrane  of  the  septum  of  the  nose  of  a cat 
or  other  animal.  The  tissue  has  been  previously  hardened  in  Muller’s 
fluid,  or  in  chromic  and  spirit  fluid,  or,  what  is  better,  a saturated 
watery  solution  of  corrosive  sublimate  for  two  or  three  hours.  In  the 
last  case,  every  trace  of  the  metallic  salt  must  be  removed  by  prolonged 
and  frequent  washing  with  alcohol.  Stain  the  section  with  logwood 
and  mount  it  in  balsam ; or  picro-carmine  can  be  used. 

(a.)  Observe  several  layers  of  cells  (fig.  102),  but  only  the  super- 
ficial layer  of  cells  is  furnished  with  cilia,  which  are  placed  on  a 
clear  disc  on  the  free  end  of  the  cell.  Notice  the  shape  of  the  cells 
in  the  subjacent  layers.  Those  of  the  lowest  layers  are  nearly 
spherical,  while  in  the  intermediate  layers  they  are  more  elongated, 


Fig.  103.  — Various  Forms  of 
Goblet-Cells  from  the  Mucous 
Membrane  of  the  Hard  Palate 
and  (Esophagus  of  the  Frog. 
One  of  the  cells  shows  mucus 
exuding  from  the  open  mouth 
of  the  cell.  Dilute  alcohol 
and  picro-carmine,  x 300. 


and  are  described  as  battledore-cells,  arranged  in  between  the  others. 
They  replace  the  ciliated  cells  when  the  latter  are  shed. 

10.  Isolated  Goblet-Cells  (H). — These  are  readily  obtained  by 
macerating  the  stomach  of  a frog  in  dilute  alcohol  for  twenty-four 
hours.  Scrape  the  surface  and  diffuse  the  cells  in  glycerine.  They 
may  be  stained  with  picro-carmine,  or  they  may  be  diffused  in  salt 
solution  and  stained  with  methyl-violet,  but  the  latter  preparation 
cannot  be  preserved  in  glycerine,  hlumerous  goblet-cells  will  be 
found  in  7. 

(a.)  Observe  the  isolated  cells  (fig.  103).  Each  cell  is  filled  for 
more  than  three-fourths  of  its  capacity  with  mucus,  while  at  the 
lower  tapering  end  there  is  a nucleus  embedded  in  a small  quantity 
of  protoplasm.  Sometimes  a plug  of  mucus  may  be  seen  exuding 
from  the  open  mouth  of  a cell. 

(b.)  With  a high  power  the  interior  of  the  upper  part  of  these 
cells  may  be  seen  to  contain  a fine  network  of  fibrils.  In  the  meshes 
is  a substance,  mucigen,  and  when  this  is  acted  on  by  water  it  yields 
mucin.  In  a certain  sense  these  bodies  are  unicellular  muciparous 
glands. 


140 


PRACTICAL  HISTOLOGY. 


[VI. 


ADDITIONAL  EXERCISES. 

11.  Cover-Glass  Preparation  of  Goblet-Cells. — Place  the  oesophagus  or 
stomach  of  a frog  in  dilute  alcohol  for  twenty-four  hours.  Scrape  the  mucous 
surface  and  compress  the  scrapings  between  two  cover-glasses.  Separate  the 
cover-glasses,  allow  the  film  adhering  to  each  glass  to  dry,  and  then  stain 
it  with  eosin  or  aniline-water-methyl-violet,  or  safranin-0.  Wash  off  the 
surplus  stain  with  absolute  alcohol,  allow  the  fdm  to  dry,  and  mount  it  in 
xylol-balsam. 

Pei  haps  a better  plan  still  is  to  stain  the  cover-glass  preparations  for 
twenty-four  hours  in  Ehrlich-Biondi  fluid.  It  is  prepared  thus  : — 


Ehrlich- Biondi  Fluid, 

Saturated  watery  solution  of  orange  . 

,,  ,,  acid  fuchsin 

,,  ,,  methyl-green  . 


loo  cc. 
20  ,, 
50  ,, 


The  solutions  used,  however,  must  be  saturated.  When  used  as  a staining 
agent,  this  strong  fluid  is  diluted  with  about  forty  volumes  of  water. 

(a.)  Observe  the  goblet-cells  with  their  characters  retained  intact.  In  the 
Ehrlich-Biondi  preparation  the  protoplasm  is  stained  red,  the  nuclei  and 
nucleoli  bluish. 

12.  Cover-Glass  Preparation  of  Ciliated  Epithelium. — The  mucous  mem- 
brane of  the  oesophagus  of  a frog  is  placed  for  twenty-four  hours  in  dilute 
alcohol.  A cover-glass  preparation  is  inade  of  the  epithelium,  and  stained  as 
described  under  11,  with  methylene-blue,  safranin-0,  gentian-violet,  or 
Ehrlich-Biondi  fluid,  and  mounted  in  xylol-balsam. 

13.  T.S.  Tongue  of  Frog(|-|). — By  means  of  hedgehog-spines,  pin  out  the 
tongue  of  a frog  on  a thin  layer  of  cork  with  a small  hole  in  it.  Harden  it  for 
two  hours  in  a saturated  watery  solution  of  corrosive  sublimate  ; remove  every 
trace  of  the  sublimate  by  prolonged  washing  in  alcohol — not  water.  Stain  in 

bulk  in  picro-carmine  or  borax- carmine. 
Make  transverse  sections — best  by  the 
paraffin  infiltration  embedding  method 
(p.  41)  — and  mount  the  sections  in 
balsam. 

{a.)  Observe  the  fibro-muscular  basis  of 
the  tongue,  covered  on  the  surface  with 
(dilated  epithelium  cells  ; between  the 
ciliated  cells  the  goblet-cells,  each  with  an 
open  mouth  and  its  plexus  of  fibrils  with 
mucigen  in  its  meshes  (fig.  104). 

(6.)  Observe,  too,  how  the  expanded 
ovoid  goblet-cells  compress  the  ciliated 
cells  and  cause  the  latter  to  have  a peculiar 
shape,  a broad  expanded  top  and  a narrow 
body. 

(c. ) The  young  cells  at  the  base  of  the 
ciliated  cells. 

If  the  specimen  be  stained  in  picro-carmine  and  mounted  in  balsam,  the 
yellow  colour  of  the  picric  acid  can  be  retained  by  putting  a little  picric  acid 
into  the  alcohol  used  to  dehydrate  it,  or  by  picric  acid  placed  in  the  clove-oil 
or  xylol  used  to  clear  up  the  section. 


Fig.  104 — V.S.  Ciliated  Epithelium  of 
Frog’s  Tongue,  m.  Muscular  fibres. 
Corrosive  sublimate  and  picro-car- 
carmine,  x 250. 


VII.] 


MITOSIS  OR  KARYOKINESIS. 


141 


LESSOR  YIL 

STRUCTURE  OP  CBLLS—MITOSIS  OR  KARYO- 
KINBSIS. 

Structure  of  the  Animal  Cell. — To  see  all  the  structures  in  an 
animal  cell  is  by  no  means  easy.  Speaking  generally,  the  tissues 
of  the  articulata,  amphibians,  and  reptiles  yield  the  largest  tissue 
elements  for  examination.  The  cells  may  he  examined  in  the  fresh 
condition  or  after  “ fixing,”  hardening,  and  staining. 

In  Fresh  Condition. — Examine  a teased  preparation  in  an 
indifferent  fluid,  e.p.,  normal  saline,  the  liquid  of  Ripart  and  Petit 
(p.  24),  or  in  solution  of  methyl-green  (p.  74).  The  preparation  may 
be  sealed  u})  with  paraffin  wax  (p.  iii)  and  examined  after  some 
time. 

After  Hardening. — The  best  osmic  acid  “fixing”  fluids  for  this 
purpose  are  the  fluids  of  Flemming,  Rahl,  and  Eol,  and  the  best 
stains  safranin  and  gentian-violet.  Mount  in  xylol-balsam. 

To  see  the  finer  details  an  oil-immersion  lens  and  Abbe’s  con- 
denser must  he  used. 

Mitosis  or  Karyokinesis. — By  these  terms  is  meant  the  remark- 
able series  of  phenomena  which  take  place  in  cells — animal  and 
vegetable — when  they  undergo  a process  of  indirect  division.  In 
this  connection  it  is  important  to  remember  the  constitution  of  a 
cell  and  some  of  the  terms  which  have  been  applied  by  different 
authors  to  its  several  parts.  A cell  may  or  may  not  possess  a dis- 
tinct cell-wall,  but  the  cell-body  appears  to  he  made  up  of  two 
substances,  which  Flemming  names  as  follows  : — One  composed  of 
threads,  seldom  forming  a network,  and  called  by  him  cyto-mitoma 
or  mitoma  (/xtro?,  thread),  also  called  spongioplasm,  and  the 
other,  homogeneous  and  lying  in  the  meshes  of  the  latter,  is  the 
paramitoma  or  hyaloplasm.  The  cell-contents  are  generally 
described  as  consisting  of  a finely-granular  soft  substance,  the  so- 
caWed  protoplasm  (fig.  105).  This  protoplasm  consists  of  a network, 
sometimes  called  a filar  mass  ” or  spongioplasm^  which  lies  em- 
bedded in  a homogeneous  ground-substance  or  “ inlerfilar  mass  ” or 
hyaloplasm.  The  filar  mass  corresponds  to  the  mitoma  of  Flemming 
and  to  the  spongioplasm  of  some  other  authors,  while  the  interfilar 
mass  corresponds  to  the  paramitoma^  or  the  hyaloplasm^  or  pjara- 
plasma  of  some  authors. 


142 


PRACTICAL  HISTOLOGY. 


[VII. 


Protoplasm. 


Nucleus 


Nuclear  Membrane. 
Nuclear  Network. 

Nucleoli. 


Fig.  105.— Connective-Tissue  Corpuscle  from 
the  Skin  of  a Salamander. 


The  nucleus  (fig.  105),  bounded  by  a nuclear  membrane,  com- 
posed of  two  layers,  an  outer  one,  Avhich  does  not  stain  (achromatic), 
and  an  inner  one,  which  does  (chromatic).  Within  the  membrane 

is  an  intranuclear  network  or 
karyomiton  (Kapvovy  a kernel) 
or  karyomitoma,  consisting  of 
a reticulum  of  threads  or  fine 
fibres,  arranged  sometimes  in 
the  form  of  a regular  network. 
As  these  threads,  or  at  least 
particles  in  them,  stain  readily 
with  certain  dyes,  e.p.,  safranin, 
they  have  been  called  chromatin 
or  composed  of  chromoplasm. 
In  the  meshes  of  this  more  or  less  perfect  network  lies  the  nuclear 
fluid,  which,  however,  does  not  stain  with  certain  pigments  : it  has 
been  called  achromatin. 

In  the  meshes  of  the  reticulum  lies  one — usually  more  than 
one — nucleolus.  It  is  more  retractile  than  the  rest  of  the  nucleus. 
Generally,  however,  as  stated,  two  or  more  nucleoli  are  present, 
and  they  seem  to  differ  in  their  chemical  constitution,  so  that 
Flemming  speaks  of  princAj)ol  and  accessory  nucleoli.  ]\Iany  of 
the  bodies  described  as  nucleoli  are  really  parts  of  the  intranuclear 
fibrillar  network  seen  in  optical  section.  Other  observers  have 
ap|)lied  different  terms  to  these  structures,  but  here  it  is  not 
necessary  to  multiply  terms.  The  “attraction  sphere”  existing  in 
the  ]irotoplasm  of  some  cells  seems  to  exercise  some  influence  on 
the  dividing  nucleus. 

The  great  majority  of  cells  reproduce  themselves  by  indirect 
cell-division  or  mitosis,  and  in  this  process  the  network  within 
the  nucleus  plays  a most  remarkable  part.  The  division  of  a cell 
is  always  preceded  by  the  division  of  the  nucleus.  Starting  from 
the  resting  nucleus,  where  the  threads  are  not  well  developed,  soon 
two  poles  appear  in  the  nucleus,  and  then  the  threads  grow 
thicker,  more  numerous  and  tortuous,  forming  the  convolution 
stage.  The  various  stages  are  indicated  in  the  following 
scheme  : — 

Mitosis. 


1.  Resting  nucleus.  The  mother  nucleus  showing  the  fibrils  in  the 

reticulum  or  network  stage. 

2.  Skein  or  spirem.  Close  skein  of  fine  convoluted  fibrils,  then  thicker 

loops  running  from  polar  to  antipolar  regions,  the 
nucleoli  disappear. 

8.  Cleavage  of  fibrils.  Each  loop  (usually  y-shaped)  splits  longitudinally 
into  two  and  the  achromatic  spindle  appears. 


143 


VII.]  MITOSIS  OR  KARYOKINESIS. 


4.  Monaster^  Star,  or  The  acliromatic  spindle  distinct  with  two  poles, 
equatorial  stage.  terminating  in  two  polar  corpuscles  (witli 
cytastcrs),  nuclear  membrane  lost.  The  V-shaped 
chromatin  fibres  arrange  themselves  in  the  equator 
of  the  spindle.  Cytoplasm  separates  into  a clear 
and  a granular  zone. 


5.  Metakinesis  or 

Divergence. 

6.  Dy aster  or 

Double  Star. 

7.  Dispirem  or 

Double  Skein. 


Sister  threads  separate  and  move  towards  poles  along 
fibres  of  the  spindle. 

Complete  separation  of  the  two  sets  of  V-shaped 
sister  thread”,  towards  the  poles  of  the  spindle. 

Open  skein  in  daughter  nuclei  passing  into  a close 
skein.  Nuclear  membrane  forming.  Cell  itself 
divides. 


8.  Network  or 

Reticulum. 


Resting  condition  of  daughter  nuclei.  Cytoplasm 
divided,  remains  of  spindle  disappear.  Chromatic 
fibres  more  twisted. 


Ill  the  preparations  one  readily  finds  examples  of  these  and  the 
other  stages  of  mitosis. 

Mitosis. — By  far  the  best  animals  to  use  for  studying  the  process 
of  mitosis  are  the  larvae  of  the  water-salamander.  The  young 


Fig.  io6.— Mitosis.  A.  Nuclear  reticulum,  resting-stap^e ; B.  Preparing  for  division;  C. 
Wreath  stage;  D.  Mou aster  with  achromatic  spindle ; E.  Barrel  or  pithode  stage  or 
metakinesis,  i.e.,  chromatin  fibrils  travelling  along  the  achromatic  spindle  towards  the 
poles  ; F.  Diaster ; G.  Daughter  wreath  stage  ; H.  Daughter  cells  passing  to  resting- 
stage. 

animals  must  he  carefully  fed,  else  the  mitotic  figures  are  not  well 
seen.  They  are  killed  at  various  stages,  when  they  vary  in  length 
from  f to  I inch  in  length.  They  may  be  hardened  in  ^ per  cent, 
chromic  acid. 

A very  good  hardening  reagent  is  one-sixth  p.c.  chromic  acid. 
From  a week  to  ten  days  is  sufficient.  If  Flemming’s  mixture  (p.  32) 
be  used,  the  tissues  must  remain  in  it  twenty-four  hours  or  less. 
Some  prefer  Kabl’s  mixture  (12-24  Fours)  (p.  31),  others  absolute 
alcohol  or  picric  acid.  After  being  “fixed”  the  tissues  are  placed  in 


144 


PRACTICAL  HISTOLOGY. 


[VII. 


30  to  50  per  cent,  alcohol,  the  alcohol  being  renewed  frequently 
until  no  colouring  matter  is  given  off.  They  can  then  be  preserved 
in  strong  alcohol,  but  on  prolonged  keeping  the  nuclei  change 
somewhat. 

1.  T.S.  Tail  of  Larva  of  Salamander  (H). — Stain  a section  for 
12-24  hours,  or  even  longer,  in  a solution  of  safranin  (p.  75) — a 
saturated  alcoholic  solution  diluted  with  half  its  volume  of  Avater. 


hydrochloric  acid).  This  rapidly  de- 
colorises it  (^-i  min.).  Instead  of  acid  alcohol  the  absolute  alcohol 
may  be  used.  It  removes  the  surplus  stain  more  slowly.  The 
difficulty  is  just  to  hit  the  moment  when  the  dye  is  washed  out  of 
the  nuclear  matrix,  the  fibrils  being  still  stained.  The  section  is 
then  transferred  to  absolute  alcohol  and  clarified,  and  mounted  in 
xylol-balsam.  The  best  agent  to  clarify  the  section  is  cedar-wood 


safranin-stained  specimens,  the  nuclear  spindle  is  not  usually  well 
seen. 

2.  Surface-Scraping  of  the  Epidermis,  Cornea,  or  External 
Gills. — Instead  of  making  a section,  scrape  the  surface  of  the  skin 
of  the  tail,  or  break  up  the  external  gills  in  water,  or  stain  (safranin) 
and  mount  the  cornea  in  xylol-balsam.  Stain  either  with  safranin 
or  logwood,  mount  in  balsam,  and  numerous  mitotic  figures  will  be 
found  (fig.  108). 


In  dealing  with  such  delicate  sections, 
it  is  well  to  ‘‘fix  ’’  the  section  on  a 
slide  beforehand,  especially  if  it  be 
cut  in  paraffin.  The  section  may  be 
fixed  by  albumin  and  glycerine,  the 
paraffin  removed  by  turpentine  or 


oil,  as  it  does  not  dissolve 
the  safranin,  which  clove-oil 
does. 


Fig.  108. —Mitotic  Figures  from  the  Epidermis 
of  a Young  Salamander.  Chromic  acid  and 
safranin,  x 300. 


(a.)  Observe  the  layers  of 
e})ithelium  of  the  epidermis 
(fig.  107).  In  several  of 
the  nuclei  the  characteristic 
mitotic  figures  are  to  be 
seen,  and  in  one  or  two 
sections  it  is  not  difficult  to 
pick  out  examples  of  nearly 
all  the  stages  of  nuclear 
division.  While  the  nuclear 
fibres  are  well  seen  in 


MITOSIS  OR  KARYOKINESIS. 


HS 


VII.] 

Some  prefer  to  stain  the  sections,— after  liardening  in  picric 
acid — with  Kleinenberg’s  logwood  diluted  with  the  alcoholic 
solution  of  alum  and  calcium  chloride  (p.  68),  allowing  the  sections 
to  stain  for  1 2 hours  or  longer.  They  are  mounted  in  balsam. 

N.B. — It  is  important  to  note  that  tissues  must  be  treated 
differently  according  as  one  wishes  to  see  the  chromatic  fibres  or  the 
achromatic  spindle.  To  see  the  ordinary  mitotic  figures,  osmic  acid, 
or  any  fluid  containing  it,  is  good  (12-24  hours) ; hut  in  order  to  see 
the  achromatic  spindle,  it  is  better  to  use  a chromo-acetic  mixture 
(p.  31)  for  12  hours. 


ADDITIONAL  EXERCISES. 

Mitosis,  however,  can  also  be  studied  in  mammalian  tissues. 

3.  Mitosis  in  Omentum  of  New-Born  Rabbit. — Orth  recommends  the 
omentum  of  a new-born  rabbit.  Harden  it  for  twenty-four  liours  in  Flem- 
ming’s fluid  (p.  25) ; wash  it  tlioroughly,  and  stain  it  in  safranin-0  ; wash  in 
water,  and  remove  the  surplus  dye,  if  necessary,  by  means  of  alcohol  acidu- 
lated with  hydrochloric  acid  (p.  144).  In  the  cells  of  the  milk-spots  and  in  the 
walls  of  the  blood-vessels  it  is  easy  to  detect  mitotic  figures,  but  they  are  much 
smaller  than  in  the  salamander. 

4.  Mitosis  in  the  Amnion. — One  of  the  rendiest  sources  is  the  amnion  of  a 
pregnant  rat,  as  recommended  by  Solger.  After  the  rat  is  killed,  the  uterus 
is  excised  and  placed  in  a saturated  watery  solution  of  picric  acid.  The  uterus 
and  the  membranes  round  each  foetus  are  opened  under  the  picric  fluid. 
Harden  for  twenty-four  hours  ; wash  well  in  water,  and  harden  in  the  various 
strengths  of  alcohol,  beginning  with  70  per  cent.  Better  results  are,  I think, 
obtained  by  removing  the  picric  acid  by  washing  in  alcohol  instead  of  water. 
Select  the  amniotic  membrane  and  tinge  a small  part  of  it  in  Ehrlich’s  acid 
h£ematoxylin  (p.  69)  diluted  one-half.  The  membrane  may  also  be  hardened 
in  Flemming’s  fluid  and  stained  with  safranin. 

5.  Method  of  Martinotti  and  Resegotti. — Small  pieces  of  the  tissue,  c.g.y  a 
rapidly  growing  tumour,  are  hardened  in  abs(diite  alcohol.  Sections  are  made 
and  coloured  in  a watery  solution  of  safranin-0.  The  decolorisation,  how- 
ever, is  obtained  by  a hydro-alcoholic  solution  of  chromic  acid.  Take  one  to 
two  parts  of  a I per  cent,  solution  of  chromic  acid  to  eight  or  nine  of  alcohol. 
After  it  is  sufficiently  decolorised — ^.c.,  the  colour  is  removed  from  every  part 
except  the  fibrils  of  the  nuclei — wash  the  section  in  absolute  alcohol,  clarify 
in  oil  of  bergamot,  and  mount  in  balsam.  This  method  yields  excellent 
results. 

6.  Mitosis  in  Plants. — Various  plants  have  been  recommended,  but  my  own 
experience  is  limited  to  the  following  : — 

{a.)  Take  the  fruit  of  Fritillaria  imperialis  they  are  30-40  mm.  in 

length,  and  place  them  in  absolute  alcohol  for  a week.  Then  in  equal  parts 
of  glycerine  and  absolute  alcohol  for  24  hours.  Then  cut  the  fruit  in  two  ; 
with  a dissecting  microscoj)e  search  for  the  embryo-sac.  It  shows  various 
stages  of  mitosis  after  staining  for  12-24  liours  in  safranin.  Mount  in  xylol- 
balsam. 

{h.)  A transverse  section  of  the  fruit  of  Lilium  candidum  also  does  very  well. 
14  K 


PRACTICAL  HISTOLOGY. 


146 


[VIII. 


Harden  the  buds  in  absolute  alcohol  when  they  are  about  i-ij  cm.  long. 
Stain  in  safranin  and  mount  in  xylol-balsam. 

(c. ) I have  also  tried  the  young  growing  shoots  of  an  onion  bulb  placed  in 
water.  The  tips  of  the  growing  rootlets  w^ere  hardened  in  Fol’s  or  Rabl’s  fluid 
and  stained  with  safranin.  Fairly  good  specimens  were  thus  obtained. 


CELLULAR  AND  HYALINE  CARTILAG-E. 

Cartilage. — The  varieties  of  cartilage  are  classified  as  follows  : — 

1.  Cellular  or  Parenchymatous  occurs  in  the  chorda  dorsalis,  ear 

of  mouse  and  rat. 

2.  Hyaline  encrusts  the  articular  ends  of  bones,  occurs  in  costal, 

tracheal,  bronchial,  laryngeal  (except  epiglottis  and  cornicula 
laryngis),  nasal  cartilages,  external  auditory  meatus,  and  in 
the  “ temporary  ” cartilages  of  the  foetus. 


(a.)  White  fibro-cartilage  occurs  in  the  intervertebral  discs, 
interarticular  fihro- cartilages,  as  marginal  cartilages  on 
the  edge  of  joints  (hip,  shoulder),  lining  tendon 
grooves,  in  sesamoid  hones,  the  sacro-iliac  synchon- 
drosis and  symphysis  pubis. 

{h.)  Yellow  fibro-cartilage  occurs  in  the  epiglottis,  cartilages 
of  Wrisberg  and  Santorini,  external  ear,  and  Eustachian 
tube. 

I.  Cellular  Cartilage  (H). — Kill  a rat  or  mouse  ; snip  off  the  ear. 

AVith  a stout  pair  of  forceps  remove  the  skin  and  the  other  tissues 


them  may  be  spherical,  but  most  are  polygonal  in 
^^^cSia^ge^^from  shape,  closely  pressed  together,  and  united  by  a very 
the  Ear  of  a small  amount  of  matrix  or  intercellular  substance  {m), 
Scohob  x^25o*^  By  focussing,  rows  of  them  in  several  planes  may  be 
seen.  Usually  no  nucleus  is  visible  in  the  cells. 

If  desired,  a section  can  be  stained  with  hsematoxylin  and 
mounted  in  balsam. 


LESSON  YIII. 


3.  Fibrous. 


{a.)  White  fibro-cartilage. 
(b.)  Yellow  fibro-cartilage. 


from  the  ear  until  the  thin  lamella  of  cartilage 
which  forms  the  basis  of  the  ear  is  exposed. 
Harden  the  cartilage  in  absolute  alcohol.  Mount  a 
thin  part  of  the  cartilage  in  Farrant’s  solution. 


1.  Cellular  Cartilage  (H). 

(a.)  Observe  the  clear  cells  (fig.  109,  c)  ; some  of 


VIII.]  CELLULAR  AND  HYALINE  CARTILAGE.  14/ 

II.  Hyaline  Cartilage.— This  consists  of  cells  or  corpuscles  em- 
bedded in  a hyaline  matrix. 

2.  Cartilage  of  Newt  (L  and  H). — Snip  otf  a small  piece  of  the 
thin  cartilage  of  the  sternum  of  a freshly-killed  newt,  and  with  a 
scalpel  scrape  away  any  fibrous  tissue  or 
muscle  adhering  to  it.  Mount  it  in  normal 
saline  solution  or  | per  cent,  solution  of  alum. 

(a.)Observe  a homogeneous  matrix  (fig.  no, 
m),  like  ground  glass,  in  which  are  embedded 
here  and  there  cartilage-cells  or  corpuscles 
(c).  The  matrix  is  comparatively  small  in 
amount,  and  hyaline. 

(h.)  Each  corpuscle  consists  of  a spherical 
mass  of  transparent,  finely-granular  cell-sub-  tt  r n 

stance  (c).  Sometimes  the  protoplasm  con-  tiiage.  m.  Matrix;  c. 
tains  retractile  granules  of  oil,  and  in  it  is 
placed  a spherical,  clear,  granular  nucleus  (?i). 

Near  the  margin  of  the  preparation  may  be  seen  cavities  or  capsules 
from  which  the  cell-contents  have  fallen  out ; others  where  the 
cell-contents  have  shrunk  from  their  capsule  ; Avhile  at  other  places 
the  cells  com])letely  fill  the  spaces  in  which  they  lie.  On  focussing 
through  the  thickness  of  the  tissue,  the  cells  are  seen  to  be  two  or 
more  layers  deep,  i.e.,  in  a section  of  moderate  thickness  they  lie  in 
several  planes.  The  cells  may  lie  singly  or  in  groups. 

3.  Effect  of  Acetic  Acid  (H). — Irrigate  with  a 2 percent,  solution 
of  acetic  acid. 

(a.)  Observe  that  the  nucleus  becomes  more  distinct  and  granular, 
the  cell-contents  clearer,  and  the  cell  shrinks  from  its  capsule,  so  that 
a space  is  left  between  the  capsule  and  the  irregular  shrunken  cell- 
contents. 

4.  Action  of  Gold  Chloride  (H). — Mount  in  Tarrant’s  solution 
a section  of  articular  cartilage  from  the  head  of  the  femur  of  a 
freshly-killed  frog  which  has  been  stained  by  the  gold  chloride 
method  (p.  78).  If  the  gold  he  reduced  by  formic  acid  the  bone  is 
thereby  softened,  so  that  both  bone  and  cartilage  can  be  cut  together 
in  a freezing  microtome. 

(a.)  Observe  the  matrix  faintly  stained,  and  the  corpuscles  or 
cell-contents,  but  not  nucleus,  stained  of  a purple  hue.  The  cell- 
contents  have  shrunk  very  little.  Here  and  there  an  empty 
cartilage-capsule  may  be  seen.  The  gold  chloride  has  a special 
affinity  for  the  protoplasm  of  the  cartilage-cells,  so  that  they  stand 
out  distinctly  in  contrast  to  the  less-stained  matrix  in  which  they 
lie  embedded.  This  preparation  represents,  as  it  were,  the  positive 
picture,”  in  contrast  to  that  obtained  by  the  use  of  silver  nitrate, 
which  yields  the  ‘‘negative  picture”  (p.  77). 


148 


PRACTICAL  HISTOLOGY. 


[VIII. 


As  a general  rule,  it  may  be  stated  that  gold  chloride  stains  the 
cellular  elements,  whilst  silver  nitrate  stains  the  cement  substance, 
e.g.^  connective  tissue,  cornea,  &c. 

5.  Costal  or  Tracheal  Cartilage  (L  and  H). — With  arazor  mak(i 
a thin  transverse  section  of  a fresh  rib  cartilage  or  tracheal  cartilage, 
e.g.^  of  a dog,  cat,  or  rabbit,  and  examine  the  section  in  normal 
saline. 

(a.)  (L)  Observe  the  circular  or  oval  outline  ^of  the  section 
surrounded  by  the  perichondrium  firmly  adherent  to  the  cartilage, 
which  consists  of  cells  embedded  in  a hyaline  matrix  (fig.  1 1 1,  Pch). 


Fig.  III. — Hyaline  Cartilage.  T.S.  Human  thyroid  hardened  in  alcohol,  x.  Fibrous 
matrix ; Chb.  Cartilage-capsules ; Pch.  Perichondrium. 

(&.)  (H)  Observe  that  the  cells  are  smaller  and  flattened  near  the 
periphery,  fusiform  farther  in,  oval  or  spherical  nearer  the  centre  of 
the  section.  They  may  lie  singly,  or  in  groups,  or  in  rows. 

(c.)  The  matrix  is  generally  hyaline,  but  in  some  places  it  may 
be  fibrous  (figs,  iii,  x,  and  112,/). 

{(L)  Around  each  cell,  or,  it  may  be,  each  group  of  cells  (fig.  112, 
3 or  2),  look  for  a car tiUig e-capsule  (fig.  iii).  It  is  firmly  united 
to  and  continuous  with  the  matrix ; but  by  tilting  the  mirror 
slightly,  so  as  to  modify  the  light,  it  may  be  seen  distinctly  as  a 


CELLULAR  AND  HYALINE  CARTILAGE. 


149 


VIII.] 

well-defined  membrane  bounding  the  cell-cavity.  Not  unfrequently 
several  so-called  ‘‘  daughter-cells”  may  be  seen  within  one  capsule. 

6.  Hardened  Costal  Cartilage. — Mount  a transverse  section  of 
costal  cartilage  which  has  been  hardened  in  a saturated  solution 
of  picric  acid.  The  picric 
acid  is  removed  by  washing 
in  alcohol. 

(a.)  Stain  a section  with 
picro-carminc  and  mount  it 
in  Tarrant’s  solution. 

(b.)  Stain  a section  in 
hoematoxylin  and  mount  it 
in  balsam.  The  matrix  is 
stained  of  a light  blue,  and 
the  corpuscles  of  a deeper 
tone. 

(c.)  A very  good  stain  for 
hyaline  cartilage  is  Merkel’s 
indigo-carmine  stain  (p.  67). 

The  preparation  can  be 
mounted  in  balsam,  and  is 
not  too  transparent. 

(d.)  Carmine.  — Place  a 
similar  section  for  twenty- 
four  hours  in  a strong  solu- 
tion of  ammoniacal  carmine 
(p.  63).  Wash  away  the 

surplus  carmine,  and  allow  a 
drop  of  strong  glacial  acetic 
acid  to  fall  on  the  section.  After  a minute  or  so,  wash  the  section 
thoroughly  in  water  to  remove  all  the  acid,  and  mount  it  in  Tarrant’s 
solution. 

Observe  the  same  arrangement  of  the  cells  as  before,  but  the 
cells  are  stained  red  while  the  matrix  is  colourless.  The  connec- 
tive-tissue corpuscles  of  the  perichondrium  are  also  red.  Hound 
each  cartilage-cell  is  a thin  outline  deeper  stained  than  the  rest, 
indicating  the  presence  of  a cartilage-capsule. 

7.  Fat  in  Cartilage-Cells  ( H). — Place  a section  of  costal  cartilage 
(preferably  made  from  a piece  of  costal  cartilage  taken  from  a 
person  over  fifty  years  of  ago)  in  i per  cent,  osniic  acid  for  an  hour, 
wash  it  in  water,  and  mount  in  Tarrant’s  solution. 

(a.)  Observe  in  some  of  the  cells  small  and  somewhat  larger 
black  spots,  which  are  globules  of  oil  blackened  by  the  osmic 
acid. 

8.  Eosin  (H). — Stain  with  a watery  solution  of  eosin  a section 


Fig.  112.— T.S.  Human  Costal  Cartilage,  z.  Cell 
shrunk  from  the  wall  of  its  cavity,  h ; i.  Two 
cells  in  one  cartilage-capsule,  k;  at  a;  is  the 
commencement  of  a separation  wall ; 2.  Five 
cartilage-cells  within  one  capsule,  hut  the 
lowest  cell  has  fallen  out  of  its  cavity ; 3.  Car- 
tilage-capsule cut  obliquely,  so  that  it  appears 
thicker  at  one  side ; 4.  Cartilage-capsule  not 
opened  into  ; g.  Hyaline  matrix ; /.  Fibrous 
matrix,  x 300. 


150 


PRACTICAL  HISTOLOGY. 


[VIII. 


Hyaline 

Cartilage. 


of  human  costal  cartilage  from  an  adult.  The  section  becomes 
uniformly  red.  Wash  it  in  dilute  acetic  acid  and  mount  it  in 
Farrant^s  sohition. 

(a.)  The  cells  are  more  deeply  stained  than  the  matrix,  and 
numerous  cells  will  be  seen  in  groups  or  in  rows  due  to  the  pro- 
liferation of  cartilage-cells.  The  cartilage-capsules  are  usually 
more  deeply  stained  than  the  surrounding  matrix.  Look  for  a 
part  of  the  matrix  which  has  Jil)rous,  It  is  deeply  stained. 

If  the  mirror  be  slightly  tilted,  or  the  light  shaded  from  the  pre- 
paration by  the  hand,  the  ‘cartilage- 
capsules  are  usually  distinctly  seen. 

9.  Articular  Cartilage.— Decalcify 
the  head  of  a long  bone  (^.<7.,  the  femur) 
of  a cat  or  other  animal  in  picric  acid 
or  chromic  and  nitric  acid,  with  the 
precautions  indicated  at  p.  37.  When 
it  is  thoroughly  decalcified,  make — by 
freezing — vertical  sections,  so  as  to  in- 
clude the  encrusting  cartilage  and  the 
subjacent  cancellous  bone.  Place  some 
sections  in  i per  cent,  osmic  acid  for 
twenty-four  hours,  and  stain  others  in 
picro-carmine.  Mount  examples  of 
both  in  glycerine-jelly,  as  glycerine  or 
Far  rant’s  solution  makes  the  tissues 
rather  too  transparent. 

(«.)  (L)  Observe  the  layer  of  en- 
crusting cartilage  fixed  upon  the  can- 
cellated bone  beneath  (fig.  113),  a 
bold,  irregular,  wavy  line  separating 
the  cartilage  from  the  bone,  but  the 
one  dovetails  into  the  other.  In  the 
cartilage  notice  two  areas,  an  upper  and 
larger  one,  wdth  a hyaline  matrix ; and 
a lower,  narrower  one,  with  a more 
granular  matrix.  The  latter  is  the  zone  of  calcified  cartilage.  A 
fine  wavy  delicate  line  indicates  where  the  hyaline  matrix  ends  and 
the  calcified  matrix  begins.  In  the  matrix  note  the  cartilage-cells, 
flattened  at  the  circumference — i.e.^  next  the  joint  cavity — in  small 
groups  deeper  down,  and  in  vertical  rows  in  the  substance  of  the 
cartilage. 

(ly.)  (H)  Study  the  shape  of  the  cells  from  the  free  or  joint  surface 
downwards.  At  the  circumference  they  are  flattened  or  fusiform,  and 
deeper  down  they  are  more  or  less  polyhedral  and  arranged  in  vertical 
rows.  Some  of  the  cells  may  be  somewhat  shrunk  within  their 


Calcified 

Cartilage. 


Bone. 


Fig.  1 13. — V.S.  Articular  Cartilage. 
Chromic  and  nitric  fluid.  Picro- 
carmine. 


viil]  cellular  and  hyaline  cartilage.  15  I 

capsules.  A row  of  cells  may  be  seen  partly  in  the  hyaline  and 
partly  in  the  calcified  matrix.  Note  the  finely 
granular  character  of  the  calcified  matrix. 

(c.)  Observe  the  bone  with  its  lamellae  and 
bone-corpuscles,  and  its  open  meshes  containing 
bone-marrow. 

10.  Cartilage  of  Cuttlefish  (H). — Alount  in 
Warrant’s  solution  a section  of  the  cephalic  car- 
tilage of  a cuttlefish.  The  cartilage  must  have 
been  hardened  previously  in  picric  acid,  alcohol, 
or  osmic  acid. 

(a.)  Stain  a section  in  picro-carmine.  Observe 
that  the  cells  lie  in  groups  of  three  or  four,  and 
from  the  periphery  of  the  group  processes  are 
given  off  which  anastomose  with  processes  from 
adjoining  groups  of  cells  (fig.  114). 

Eosin  and  Haematoxylin. — Stain  a section  . . „ 

slightly  with  a dilute  solution  of  eosin  and  after- 
wards  with  dilute  haematoxylin.  Mount  in 
Tarrant’s  solution.  The  matrix  is  reddish,  and  the  cells  and  their 
processes  purplish  in  hue. 


Fig.  114.  — Brancheii 
Cartilage-Cells  of  the 
Cartilage  of  Loligo. 


ADDITIONAL  EXERCISE. 

11.  Silver  Nitrate  and  Cartilage  Matrix  (H). — Rub  a piece  of  solid  silver 
nitrate  upon  the  cartilaginous  end  of  the  freshly-excised  femur  of  a frog. 
Expose  the  cartilage  in  water  to  sunlight.  It  rapidly  becomes  brown.  Make 
a thin  surface  section  with  a razor,  and  mount  it  in  Farrant’s  solution. 

(a.)  Observe  the  matrix  stained  brown,  and  a large  number  of  unstained 
spaces  apparently  empty.  The  latter  are  the  cavities  in  which  the  cells  lie, 
but  the  cells  themselves  are  too  transparent  to  be  readily  seen.  This  picture 
is  the  reverse  of  that  obtained  with  gold  chloride  (p.  147). 


LESSON  IX. 

THE  PIBRO-CARTILAGBS  (WHITE  AND  YELLOW). 

III.  Fibro-Cartilages. 

A.  White  Fibro-Cartilage. 

1.  Intervertebral  Disc. — Decalcify  in  chromic  and  nitric  acid 
fluid  (p.  37)  or  picro-sulphuric  acid  (24  hours)  an  intervertebral  disc 


T52 


PRACTICAL  HISTOLOGY. 


[IX. 


and  its  adjacent  pieces  of  bone  (rabbit  or  cat).  By  freezing  make 
vertical  sections  bo  include  the  disc  and  its  adjacent  bones;  place 
the  sections  for  twenty-four  hours  in  i per  cent,  osmic  acid,  wash  them 
thoroughly  in  water,  and  mount  in  Farrant’s  solution  or  glycerine- 

jelly. 

(a.)  (L)  Observe  the  decalcified  hones,  and  between  them  the 
disc  (fig.  115).  The  bone  consists,  for  the  most  part,  of  cancellated 
bone,  and  the  disc  stretches  between  the  two  plates  of  denser  bone 
which  cover  the  ends  of  the  vertebrae.  A thin  layer  of  hyaline 
cartilage  exists  on  the  surface  of  the  bodies  of  the  vertebrae. 

(b,)  The  disc  itself  is  of  considerable  thickness,  and  consists  of 
many  parallel  bundles,  between  which,  and  at  right  angles  to  them, 

are  other  bundles  cut 
transversely,  the  fibres 
of  adjoining  bundles 
being  arranged  some- 
times in  a zigzag 
fashion  (fig.  115).  In 
these  bundles  are  carti- 
lage-cells, more  numer- 
ous in  the  central 
bundles  and  fewer  in 
the  outer  ones.  In 
the  centre  of  the  disc 
may  be  seen  a more 
pulpy  tissue,  the  re- 
mains of  the  chorda 
dorsalis. 

(c.)  Externally  on 
both  sides  is  a ligament 
of  connective  tissue 
which  passes  from  one  vertebra  to  the  other.  It  gradually  shades 
into  the  fibres  of  the  disc. 

(d.)  (H)  Observe  the  fibres,  with  a greater  or  less  number  of  large 
oval  cells  lying  between  them.  The  fibres  can  perhaps  be  traced 
into  the  matrix  of  the  bone. 

(e.)  The  cells — oval  and  with  a liyaline  capsule — are  most  numer- 
ous in  the  central  part  of  the  disc,  and  usually  there  is  no  difficulty 
in  seeing  groups  of  cells  in  the  hyaline  cartilage  forming  a thin 
coating  on  the  bone.  The  boundary-line  between  the  disc  and  the 
bone  is  never  straight,  but  wavy.  This  can  readily  be  made  out  by 
tilting  the  mirror  slightly. 

2.  White  Fibro-Cartilage  (H). — Snip  off  a small  piece  of  the 
intervertebral  disc  of  an  ox  or  sheep  or  man,  after  hardening  a small 
portion  for  a day  in  a saturated  solution  of  picric  acid  or  spirit,  or 


Fig.  1 15  — V.S.  Intervertebral  Disc  of  Cat.  B.  Bone  ; D. 
Disc.  Chromo-nitric  acid  fluid  and  osmic  acid,  x 15. 


IX.] 


THE  FIBRO-CARTILAGES. 


153 


FlO. 


16. — From  Human  Intervertebral  Disc. 
m.  Matrix  or  fibrous  ground-substance ; 
c.  Cartilage-cell ; k.  Capsule  surrounded 
with  calcareous  particles.  Kfeinenberg’s 
fluid  and  borax-carmine,  x 250. 


picro-sulpliuric  acid  (twenty-four  hours).  The  hardening  is  com- 
pleted in  the  various  strengths  of  alcohol.  Tease  it  with  needles 
in  Tarrant’s  solution. 

[a)  Observe  the  fibrous  matrix,  consisting  of  very  fine,  wavy 
unbranched  fibrils  (fig.  116),  and  between  them  oval  or  spherical 
nucleated  cells,  each  one  with  a 
distinct  thick  hyaline  capsule. 

In  some  of  the  latter,  concentric 
rings  indicating  the  deposition  of 
successive  capsules,  may  be  seen. 

[h.)  The  bundles  of  fibres  run 
in  various  directions,  and  each 
fibril  is  unbranched.  The  cells 
— with  thick  capsules — are  not 
very  numerous,  and  lie  either 
singly  or  in  groups  of  two  or 
three  between  the  fibres. 

3.  Another  Method.  — The 
cells  of  this  cartilage  do  not 
stain  very  readily,  but  the  follow- 
ing method  gives  good  results  : 

— Harden  the  cartilage — small 
pieces — for  a day  or  so  in 
Kleinenberg’s  fluid  (p.  30),  and  after  washing  the  pieces  free  from 

the  picro-sulphuric  acid,  place  them  for  several  days  in  borax- 
carmine,  and  stain  them  en  masse.  Concentrate  the  pigment  in 
the  cells  by  placing  the  pieces  in  acid  alcohol  for  twenty-four  hours 
(^p.  65).  Sections  can  be  made  either  by  freezing  or  embedding  in 
paraffia.  The  cells  are  stained  bright  red. 

B.  Yellow  Fibro-Cartilage. — (i.)  Harden  the  epiglottis  of  a sheep, 
dog,  or  cat  for  forty-eight  hours  in  absolute  alcohol. 

(ii.)  Harden  a part  of  the  ear  of  a pig  in  a saturated  solution  of 
picric  acid  for  twenty-four  hours.  Wash  away  the  picric  acid  with 
alcohol,  and  in  the  various  strengths  of  alcohol  complete  the 
hardening. 

By  freezing  make  sections  of  the  epiglottis  and  ear. 

4.  Epiglottis. — Stain  a section  in  picro-carmine  and  mount  it  in 
Tarrant’s  solution. 

{a.)  (L)  Heglecting  the  stratified  epithelium  and  glands  which 
are  present,  observe  the  perichondrium  (fig.  1 1 7,  c,  /),  embracing  the 
mass  of  cartilage,  and  firmly  adherent  to  the  latter,  which  has  a 
fibrous,  yellow-stained  matrix,  studded  with  cells— stained  red — 
embedded  in  it.  The  mass  of  cartilage  may  appear  to  be  interrupted, 
or  it  may  even  be  perforated. 

(&.)  (H)  Observe  the  perichondrium,  composed  of  connective 
15 


PRACTICAL  HISTOLOGY. 


[IX. 


1 54 


..JCO 


tissue,  with  numerous  elastic  fibres ; the  latter  can  be  traced  into, 
and  become  continuous  with,  the  elastic  fibres  of  the  matrix  (Kg. 
117,  e,  /).  The  matrix  consists  of  fine  branched  and  anastomosing 
fibres  of  elastic  tissue,  stained  yellow  with  picric  acid.  Where  the 
fibres  are  cut  transversely,  they  appear  as  yellow  dots  or  granules. 

In  addition  to  these,  however,  there 
are  numerous  granules  of  elastin 
scattered  in  the  matrix.  In  this 
meshwork  notice  the  nucleated 
cells  stained  red.  Each  cell  has  a 
capsule,  but  near  the  perichondrium 
they  are  smaller  and  flattened  (^), 
while  in  the  substance  of  the  carti- 
lage they  are  larger,  oval,  or 
spherical  (?’). 

5.  Acid  Fuchsin  Method. — Stain 
a section  with  a watery  solution  of 
acid  fuchsin.  Wash  the  section  for 
a long  time  in  absolute  alcohol  and 
mount  in  balsam.  The  network  is 
intensely  red,  and  the  other  parts 
uncoloured. 

6.  Double  Staining  of  the  Epi- 
glottis.— (i.)  Stain  a section  with 
picro-carmine,  and  then  faintly  with 
logwood.  Mount  in  balsam.  To 
preserve  the  yellow  colour  of  the 
fibrils,  the  clove-oil,  with  which  the 
section  is  cleared  up,  must  be  made 
yellowish  by  dissolving  in  it  a little 
picric  acid.  The  fibres  are  yellow, 
the  cells  red,  and  the  nuclei 
purplish. 

(ii.)  Stain  another  section  with 
dilute  eosin-haematoxylin,  and  mount 
it  in  Farrant^s  solution  or  balsam. 
The  cells  take  the  logwood  tint,  and 
the  fibres  the  colour  of  eosin. 

7.  Ear  of  Pig  or  Horse. — Stain 
a section  in  picro-carmine,  and  mount  it  in  Farrant’s  solution. 

(L  and  H)  Observe  the  skin,  its  glands  and  muscles.  iN'eglect 
these,  and  note  the  perichondrium  enclosing  the  cartilage  with  a 
characteristic  arrangement  of  the  cells.  The  cells  near  the  surface 
are  small,  flattened,  and  parallel  to  it,  while  those  in  the  centre 
are  larger  and  arranged  across  the  long  axis  of  the  section. 


Fig.  117— T S.  Epiglottis  of  a Dog.  a. 
Fat-cells  in  perichondrium,  c ; e. 
Elastic  fibres ; i.  Superficial  layers  of 
smaller  cells ; r.  Layer  of  larger 
cells  with  elastic  grannies,  l.\  f. 
Perichondrium.  Alcohol  and  picro- 
carmine. 


IX.]  THE  FIBRO-CARTILAGES,  155 


Between  the  cells  is  a matrix,  wliicli  may  be  partly  hyaline  and 
partly  yellow  fibrous. 

8.  Transition  of  Hyalino  to  Elastic  Cartilage  (H). — Dissect 
out  the  arytenoid  cartilage  of  an  ox  or  sheep.  Harden  and  pre- 
serve it  in  alcohol.  Cut  sections  through  the  part  where  the 
hyaline  cartilage  merges  into  the  elastic  variety.  This  is  quite 
visible  to  the  naked  eye,  the  elastic  part  being  more  opaque  and 
yellowish  white  in  tint.  Stain  a section  with  picro-carmine,  and 


Fig.  ii8. — Elastic  Cartilage  Ear  of  Horse, 
hardened  in  alcohol.  El.  Elastic  fibres  cut 
in  various  directions ; K.  Xucleus  of 
cartilage-cell ; Knh.  Contour  of  cartilage-cell 
cavity ; Knh'.  Empty  cartilage  capsule  ; Knz. 
Cartilage-cell. 


Fig.  1 19. — Elastio  Cartilage  developing  in 
Hyaline  Cartilage  in  Arytenoid  Carti- 
lage of  a Calf,  X 100.  The  clear  spaces 
indicate  the  position  of  the  cells,  the 
shadow  part  the  hyaline  matrix. 


mount  it  in  Farrant’s  solution.  On  making  a section  of  such  a 
cartilage  in  the  fresh  condition,  one  part  has  the  pale-bluish  colour 
of  hyaline  cartilage,  and  the  other  part  is  very  faintly  yellow. 

(a.)  At  one  part  observe  hyaline  cartilage,  whose  matrix 
gradually  becomes  fibrous.  At  first  only  a few  scattered  granules 
of  elastin  are  seen,  then  the  hyaline  matrix  is  traversed  by  elastic 
fibres,  and  gradually  the  matrix  loses  its  hyaline  character,  and 
becomes  distinctly  fibrous.  Around  each  cell  there  is  a clear  area 
— hyaline — devoid  of  fibres  (fig.  1 1 9). 


156 


PRACTICAL  HISTOLOGY. 


[X. 


LESSON  X. 

CONNECTIVE  TISSUE. 


The  group  of  Connective  Tissues  includes  cartilage,  ordinal^ 
connective  tissue  (with  adipose  tissue),  adenoid  or  retiform  tissue, 
mucous  tissue,  bone  and  dentine,  (i)  These  all  subserve  more  or 
less  mechanical  functions  in  the  organism;  (2)  they  all  have  much 
in  common  in  structure,  lc.,  they  are  composed  of  cells,  and  an 
intercellular  matrix,  hut  usually  the  development  of  the  matrix 
exceeds  that  of  the  cells;  and  (3)  they  are  all  developed  from  the 
mesohlast  of  the  embryo. 


ORDINARY  CONNECTIVE  TISSUE. 


It  consists  of  the  following  structural  elements  : — 


A,  Structural  Elements. 


Fibres 


■{ 


Vhite  or  gelatinous. 
Yellow  or  elastic. 


05 

^ ■ 


f fl.  Flattened  or  lamellar  cells,  called  also  fixed  oon- 

I I nective-tissne  corpuscles, 

i Fixed  . . -{  2.  Granular  cells  (eosinopliilous  cells  ?). 

I 3.  Vacuolated  or  plasma  cells  of  Waldeyer. 

I L Clasmatocytes  of  Ranvier  (?). 

L Migratory  . Wandering  cells  or  leucocytes. 


B.  Arrangement  qf  these  Elements. 

{a.)  Areolar,  e.g.,  subcutaneous  and  submucous  tissues. 
ih.)  Bundles  in  parallel  groups,  e.g.^  tendon  (with  parallel  fibres)  and 
fascise  (fibres  crossing  at  light  angles). 

(c.)  Fenestrated  fibrous  membranes,  e.^.,  omentum. 

{d.)  Compact  bundles  crossing  in  all  dire(;tions,  e.g.^  skin. 


The  lamellar  cells  are  flattened  or  winged  plates  which  lie  on 
the  bundles  of  fibrils.  They  have  a large  oval  nucleus  lying  in  a 
clear  plate. 

The  granular  cells,  or  “ Mastzellen  ’’  of  Ehrlich,  are  often  found 
near  blood-vessels,  and  in  the  fat  present  in  areolar  tissue,  in 
the  submucous  tissue  of  the  intestine,  and  in  Glisson’s  capsule. 
The^cells  are  often  spherical,  and  the  graindes  are  numerous  and 
proteid  in  nature,  and  stain  with  aniline  dyes,  e.g.^  eosin,  hence 
the  term  sometimes  applied  to  them  “ eosinophilous  cells.” 

The  plasma  cells  were  formerly  confused  with  the  foregoing, 


X.] 


CONNECTIVE  TISSUE. 


157 


but  in  the  plasma  cells  the  protoplasm  is  vacuolated,  and  the 
vacuoles  contain  fluid.  They  sometimes  have  short  processes. 
What  the  relation  of  the  clasmatocytes  to  these  other  cells  may  be 
is  so  far  not  determined ; nor,  indeed,  do  we  know  the  relation 
between  the  granular  and  the  plasma  cells. 

The  migratory  cells  are  identical  with  the  white  blood-cor- 
puscles or  lymph-corpuscles,  and  may  therefore  be  regarded  as  an 
adventitious  element. 

Yellow  or  Elastic  Fibres  occur  in  the  ligamentum  nuchse  of 
animals  (large  fibres) ; lig.  subflava ; stylo-hyoid  ligament ; con- 
nective tissue  generally ; in  the  walls  of  the  air-tubes  and  lungs ; 
the  larger  blood-vessels,  especially  arteries ; the  vocal  cords  and 
some  ligaments  of  the  larynx ; many  organs,  e.y.,  spleen. 

1.  Yellow  or  Elastic  Fibres — Thick  Fibres  (H). — Tease  out 
in  Avater  a fragment  of  the  ligamentum  nuchse  of  an  ox;  cover 
and  examine  it.  It  can  be  mounted  in  Farrant’s  solution. 

(a.)  Observe  the  broad 
fibres  with  a definite 
outline,  yellow  in  colour, 
refracting  the  light 
strongly,  branching  and 
anastomosing,  and  some- 
times curling  at  their 
ends  where  they  are 
broken  across  (fig.  120, 

/).  A small  quantity 
of  Avhite  fibrous  tissue 
will  be  found  between 
and  supporting  the 
fibres  (h). 

(h.)  Measure  the  size 
of  one  of  the  larger 
fibres.  They  are  about 
778  /X  inch)  in 

diameter. 

(c.)  Irrigate  Avith  acetic 
acid.  The  fibres  are  not  affected,  and  no  nuclei  are  revealed  in 
them.  They  consist  of  the  substance  elastin,  Avhich  is  unaffected 
by  acetic  acid. 

Make  longitudinal  and  transverse  sections  of  the  ligamentum 
nuchse  (hardened  in  alcohol).  Stain  both  in  picro-carmine  and 
mount  in  Farrant’s  solution.  The  connective  tissue  is  thereby 
stained  red,  and  the  elastic  fibres  yelloAv. 

2.  L.  S.  Ligamentum  Nuchse  (H). — Observe  the  fibres  (yelloAvish), 
with  a small  amount  of  connective  tissue  (red)  betAveen  them.  The 


Fig.  120.—/.  Elastic  fibres  from  the  ligamentum 
nuchae  ; b.  Fine  white  fibrous  tissue,  x 300. 


PRACTICAL  HISTOLOGY. 


[X. 


158 


fibres  are  broad  with  well-defined  margins,  have  a feeble  yellow 
tint,  and  are  transparent.  They  branch  and  anastomose,  and  where 
ruptured  curl  up  at  their  ends. 

3.  T.S.  Ligamentum  Nuchae  of  Ox  (H). — Observe  the  polygonal 
ends  of  the  broad  fibres — yellow — and  nearly  as  broad  as,  or  broader 
than,  a coloured  blood-corpuscle-,  sometimes 
single,  mostly  in  groups  of  three  or  more 
(fig.  1 2 1,  a) — homogeneous  throughout.  A 
small  amount  of  connective  tissue  (c)  (red) 
between  the  groups. 

4.  Another  section  may  be  stained  with 
a watery  solution  of  magenta  and  mounted 
in  Farrant’s  solution.  The  fibres  are  stained 
Nuch*'^('ox.^‘®aTias“t™  ^ed,  but  the  pigment  is  apt  to  diffuse  into 
fibres  ; c.  Connective-tissue  the  Farrailt’s  solution, 
between  them?  n Nuclei  k a it  • i i 

of  connective-tissue  cor-  0.  A good  plan  IS  alter  hardening  the 

puscies.  Alcohol  and  borax-  ligamentum  nuchae  in  alcohol  to  stain  it  in 
carmine,  x 300.  . ^ . » , , . , , 

borax-carmine  for  several  days,  with  the 
precautions  stated  at  p.  65.  Transverse  sections  show  the  white 
fibrous  tissue  between  the  elastic  fibres,  with  its  nuclei  stained  red 
(fig.  121,  w). 

6.  Fine  Yellow  Elastic  Fibres  (H). — Harden  the  mesocolon  or 
mesentery  of  a young  rabbit  in  Flemming’s  fluid,  and  stain  it  in 
methyl-violet  as  directed  under  Lesson  X.  14, 
or  stain  it  with  magenta,  when  the  elastic 
fibres  are  stained  red ; or  with  safranin  after 
hardening  in  chromic  acid. 

(a.)  Observe  the  network  of  fine  elastic 
fibres.  Many  of  the  fibres  have  a diameter 
equal  to  one-sixth,  or  less,  of  that  of  a 


Fig.  122. — Fine  Network  of 
Elastic  Fibres  from  the 
Mesocolon  of  Rabbit. 
Flemming’s  fiuid  and 
safranin. 


coloured  blood-corpuscle  (r  /x  or 


in  diameter).  The  fibres  branch  and  anasto- 
mose, and  by  carefully  focussing,  one  can 
observe  that  the  fibres  do  not  all  lie  in 
the  same  plane  (fig.  122). 

7.  Fenestrated  Membranes  (H). — Sometimes  the  elastin  is  so 
arranged  as  to  form  sheets  or  plates  of  elastic  tissue,  e,g.,  in  the 
large  arteries ; at  other  times  these  are  perforated  with  holes,  and 
are  called  fenestrated  elastic  membranes. 

With  a pair  of  forceps  tear  off  a little  of  the  endocardium  from  a 
sheep’s  heart,  spread  it  on  a slide,  and  treat  it  with  caustic  potash. 
Or  use  the  basilar  artery,  slit  it  up  and  scrape  away  the  outer  coats, 
and  use  caustic  potash  as  before. 

(a.)  Observe  near  the  margin  of  the  preparation  the  elastic 
membrane  with  holes  in  it  (tig.  123). 


CONNECTIVE  TISSUE. 


159 


X.] 


8.  White  Fibres  of  Areolar  Tissue  (H). — Dissect  off  a thin 
lamella  from  an  intermuscular  septum,  or  remove  a little  of  the 
subcutaneous  tissue  of  a rabbit 
or  rat.  Place  it  on  a dry  slide, 
and  rapidly  spread  it  out  into  a 
thin  film,  but  do  not  let  it 
become  dry,  which  can  easily  be 
avoided  by  breathing  on  the 
preparation.  This  is  known  as 
the  “half-drying”  or  “semi- 
desiccation method,”  and  is  a 
very  useful  one,  especially  for 
sections  containing  much  con- 
nective-tissue. Place  a drop  of 
normal  saline  solution  on  the 

cover-glass  and  apply  it  to  the  Human  endocardium.  Fresh  and 

XX*/  caustic  potash. 

preparation. 

(a.)  Observe  the  unbranched  white  fibres,  wavy  in  their  course, 


Bdgf  Bdgf  Bdgf  Lc  Lc 


Fig.  124. — Areolar  Tissue  from  Intermuscular  Tissue  of  a Calf,  x 200.  Bdgf.  Connective- 
tissue  fibres,  i.e.,  bundles  of  fibres  ; Bdgz.  Connective-tissue  cells ; EIF.  Elastic  fibres  ; 
Lc.  Leucocytes. 


with  a faint,  ill-defined  outline,  crossing  each  other  in  various 
directions.  They  are  colourless,  of  feeble  refractive  power,  and 


i6o 


PRACTICAL  HISTOLOGY. 


[X. 


transparent.  The  fibres  are  striated  longitudxxially,  and  are  seen  to 
be  made  up  of  excessively  delicate  fine  unbranched  fibrils.  The 
fibres  vary  from  6 /x  to  8 /x  in  diameter  (4yV^“‘3tVo  inch).  They 
may  be  round  or  flattened,  and  are  of  indefinite  length.  Amongst 
the  white  fibres  may  be  seen  a few  fine  elastic  fibres  (EIF),  recog- 
nised l)y  their  sharper  contour,  and  by  the  fact  that  they  branch 
and  anastomose. . They  run  between,  but  never  in  the  white  fibres. 
It  is  not  often  that  the  corpuscles  are  visible  without  the  action  of 
special  reagents.  The  corpuscles  are  best  seen  in  young  animals 
(Bg.  124,  Bdgz).. 

(h.)  Irrigate  with  a 2 per  cent,  solution  of  glacial  acetic  acid; 
observe  that  the  white  fibres  swell  up,  become  clear,  gelatinous, 

and  homogeneous ; the  elastic  fibres 
being  unaffected,  come  clearly  into 
view.  The  latter  have  a sharply- 
defined  outline,  branch  and  anasto- 
mose, and  sometimes  curl  at  the  ends. 

The  corpuscles,  or  at  least  their 
nuclei,  come  into  view.  Observe 
the  oval  or  fusiform  nuclei  of  the 
fixed  connective  - tissue  corpuscles 
(Bdgz) — they  may  be  surrounded 
with  some  soft  protoplasm — and 
the  much  smaller  compound  nuclei 
of  the  wandering  cells  or  leucocytes 
(Lc).  In  the  rat  especially  one  is 
very  likely  to  find  the  very  granular 
nucleated  cells  known  as  granular 
cells.  They  frequently  lie  along 
E',  E".  Paj;-  the  course  of  the  small  blood- 
vessels. Do  not  preserve  this 
specimen. 

If  the  areolar  tissue  be  taken  from  the  sub-arachnoid  space  of 
the  brain  and  treated  with  dilute  acetic  acid,  the  fibres  lose  their 
fibrillated  structure,  nuclei  appear,  and  the  fibres  themselves  may 
be  seen  to  swell  up  here  and  there,  while  they  are  constricted  at 
irregular  intervals  by  a thin  fibre.  This  is  due  to  these  fibres 
being  partly  embraced  by  connective-tissue  cells,  which  have  long 
branches  ^rhich  ] artly  encircle  the  fibre. 

9.  Fenestrated  Fibrous  Tissue  (L 
omentum  of  a dog  or  cat  in  Muller’s  fluid, 
and  mount  in  balsam. 

Note  the  meshes  bounded  by  areolar  tissue.  The  fibrils  which 
compose  the  fibres  are  readily  seen,  and  sometimes  an  endothelial 
cell  may  be  seen  partially  detached  (fig.  125,  E'),  for  the  omentum 


Fig.  125.— Omentum  of  Dog, 

tially  detached  endothelial  cells.  E. 
Nuclei  of  endothelial  cells,  x 130. 


and  H).  — Harden  the 
Stain  a piece  in  logwood. 


X.] 


CONNECTIVE  TISSUE. 


l6l 


lying  in  a serous  cavity  is  covered  with  endothelium.  The  nuclei  on 
the  surface  are  the  nuclei  of  endothelial  cells,  and  those  in  the  sub- 
stances of  the  trabeculae  belong  to  connective-tissue  cells. 


ADDITIONAL  EXERCISES. 


10.  Martinotti’s  Reaction  for  Elastic  Fibres  ( H ). — Harden  elastic  tissue, 
e.g.,  ligamentum  nuchse,  or  an  organ  containing  elastic  fibres,  e.g.^  skin, 
artery,  lung,  trachea,  in  .2  per  cent,  chromic  acid  for  three/  weeks.  Cut 
sections  and  place  them  for  twenty-four  to  forty -eight  hours  in  a saturated 
alcoholic  solution  of  safranin-0.  Wash  them  in  acid  alcohol  (p.  65),  and  then 
in  absolute  alcohol,  to  remove  the  surplus  dye  ; 
clear  in  xylol,  and  mount  in  xylol-balsam. 

{a.)  All  the  elastic  fibres,  and  they  alone,  are  now 
either  purplish,  or,  if  the  fiiues  be  fine,  black. 

This  is  a most  excellent  method  for  differentiating 
elastic  fibres.  The  one  thing  of  importance  is  to 
secure  a good  sample  of  safranin  ; some  samples  are 
quite  inactive. 

11.  Elastic  Fibres  {Herxheimer^ s Method). — Place 
the  sections  containing  elastic  fibres  in  an  alcoholic 
solution  of  hiematoxylin,  to  which  is  added  a few 
drops  of  a saturated  solution  of  lithium  carbonate. 

Stain  them  for  a few  minutes.  Place  them  for  five 
to  twenty  seconds  in  tincture  of  perchloride  of  iron, 
which  rapidly  decolorises  all  except  the  elastic 
fibres,  which  remain  bluish  or  blackish.  Wash  in 
water  and  mount  in  balsam.  This  method  is  admir- 
ably adapted  for  demonstrating  the  longitudinal 
layer  of  elastic  fibres  in  the  trachea  and  bronchi. 

12.  Violet-B  Method. — Cut  out  the  hyaloid  mem- 
brane of  a frog’s  eye,  or  a piece  of  the  omentum  of  a 
young  rabbit,  or  the  sus{)ensory  ligament  of  the  liver 
of  a rabbit.  In  normal  saline  pencil  away  the  epi- 
thelium covering  the  membrane.  Stain  the  section 
with  violet-B  (i  gram  violet- B in  300  cc.  of  normal 
saline).  This  stains  the  cells  and  the  elastic  fibres. 

The  preparation  cannot  be  mounted  in  glycerine 
or  Farrant’s  solution,  as  these  dissolve  out  the  dye, 

but  a strong  solution  of  common  salt  may  be  used 

{S.  Mayer). 

13.  Areolar  Tissue — Permanent  Preparations. 

(i.)  By  means  of  a hypodermic  syringe  (fig.  126) 

make  an  interstitial  injection  of  silver  nitrate 
(i  : 1000)  into  the  subcutaneous  tissue  of  a dog  or 
l abbit.  In  this  way  an  artificial  oedema  is  produced 
and  the  tissues  are  “ fixed.”  With  a pair  of  scissors 
curved  on  the  flat,  snip  out  a little  of  the  now  oedematous  connective  tissue 
and  stain  it  with  picro-carmine.  It  requires  some  time  to  stain,  and  the 
preparation  should  be  left  for  ten  to  twelve  hours  in  a moist  chamber 

(fig.  70),  and  then  the  picro-carmine  is  slowly  displaced  by  acid  glycerine, 

I.C.,  glycerine  slightly  acidulated  with  formic  acid.  In  this  way  the 


Fig.  126. — Hypodermic  Sy- 
ringe for  making  a Sub- 
cutaneous or  Interstitial 
Iniection. 


PRACTICAL  HISTOLOGY. 


162 


[X. 


vaiious  elements — fibrous  and  cellular — are  usually  brought  distinctly  into 

view  {Ranvier). 

(ii.)  An  excellent  plan  is  to  inject  picro-cannine  interstitially,  and  to  leave 
tlie  bulla  several  hours  befoi-e  snipping  out  a small  part  of  it  and  mounting 
it  in  formic  glycerine.  In  this  preparation  connective-tissue  fibres  with  con- 
strictions at  intervals  are  frequently  seen. 

(iii.)  A fibre  may  be  stained  with  acid  hsematoxylin  and  mounted  in  gly- 
cerine (p.  69). 

14.  Coarsely  Granular  Cells  Mastzellen''  of  Ehrlich).— part  of  the 
omentum  of  a young  rabbit — or  the  fat  from  around  the  kidney  of  a rat  or 
rabbit — in  a watery  solution'  of  gentian-violet,  to  which  a filtered  watery  solu- 
tion of  aniline-oil  has  been  added.  Heat  the 
whole  in  a capsule  until  the  vapour  begins  to 
rise,  and  allow  it  to  cool. 

After  staining  for  twenty-four  hours,  remove  the 
tissue  and  wash,  it  in  acid  alcohol  until  most  of  the 
blue  is  gone.  Dehydrate  it  in  absolute  alcohol, 
clear  with  xylol,  and  mount  it  in  xylol-balsam. 

(H)  Search  for  a blood-vessel,  and  along  its 
course  will  be  found  large  oval  cells  crowded 
with  numerous  granules  stained  blue  (fig.  127). 
These  cells  are  found  also  apart  from  the  blood- 
vessels. 

15.  Gentian- Violet  and  Carmine  Preparation. 

— The  preparation  may  be  double  stained,  thus  : 
After  washing  in  acid  alcohol,  staiii  the  prepara- 
FiG.  127.— Coarsely  Granular  tion  in  lithium-carmine  for  a few  minutes,  and 
again  extract  with  acid  alcohol.  Mount  as  before 
/.  Fat  cells;  v.  Vein;  m.  m balsam.  Observe  the  granules  of  the  cells, 
“ Mastzellen.’  blue  as  before,  the  nucleus  red.  All  the  other 

nuclei  in  the  field  are  now  red. 

16.  Clasmatocytes. — Ranvier^  has  given  the  name  clasmatocyte  (/cAairaa, 
fragment,  kotos,  cell)  to  cells  which  can  be  seen  in  thin  connective-tissue  mem- 
branes of  vertebrates.  In  Triton  cristatus  these  cells  may  be  I mm.  in  length. 
Stretch  a membrane,  e.g.,  the  omentum  of  a mammal  or  mesentery  of  a small 
re{)tile  on  a slide.  Fix  its  elements  by  dropping  on  it  a drop  of  i per  cent, 
osmic  acid,  and  then  stain  it  with  methyl-violet-BBBBB  (i  part)  dissolved 
in  distilled  water  (10  parts).  Examine  the  preparation  in  the  fluid  or  in 
water.  Large  branched  cells  are  seen  stained  of 
a bluish  tint.  At  the  extremities  of  the  branches 
are  small  islands  or  granulations  similarly  tinted, 
and  it  is  for  this  reason  Ranvier  has  given  them 
this  name. 

17.  Cell-Spaces  in  Areolar  Tissue  (H).  — From 
a freshly-killed  rabbit  snip  out  a small  piece  of 
the  subcutaneous  tissue  as  free  from  fat  as  pos- 
sible. Spread  it  upon  a dry  slide,  and  drop  on  it 
Fig.  I28.-Cell-Spaces  in  Areo-  a,  pipette  a half  )>er  cent,  solution  of  silver 

lar  Tissue.  Silver  nitrate,  nitrate.  Allow  the  silver  to  act  for  ten  to  twelve 
minutes,  remove  it,  cover  the  film  with  glycerine 
and  expose  it  to  light.  It  rapidly  becomes  brown.  It  is  better  to  use  con- 
nective-tissue from  a calf,  but  the  layer  used  must  not  be  too  thin. 

(a.)  If  successful,  note  a brownish  ground — the  cement  substance — and  in  it 
clear  branched  spaces  corresponding  in  shape  to  the  fixed  connective-tissue  cor- 
puscles. These  are  the  cell-spaces  (fig.  128). 

^ Comptes  Rendus,  vol.  110,  p.  165,  1890. 


XL] 


TENDON. 


163 


18.  Perioesophageal  Membrane  of  Frog. —To  Ranvier^  we  owe  our  know- 
ledge of  the  value  of  this  membrane.  The  oesophagus  is  sun'ounded  by  a lyiriph- 
sac,  which  is  separated  from  the  pleuro-])eritou(‘al  cavity  by  an  excessively  thin 
membrane.  The  sac  is  readily  distended  by  insufflation,  after  pulling  out  the 
oesophagus  through  the  mouth  by  means  of  a hook.  This  membrane  is  covered 
on  both  surfaces  hy  epithelium  which  can  be  stained  with  silver  nitrate  ; its 
texture  consists  of  fine  connective-tissue  with  elastic  fibres,  but  containing 
clasmatocytes,  and  in  it  are  also  to  be  found  blood-vessels  and  non-medullated 
nerves.  Treat  it  as  de.>cribed  in  16,  to  see  the  clasmatocytes.  By  the  same  }>ro- 
cess  the  non-nn  dullated  nerve  fibres  and  elastic  fibres  will  also  be  stained. 


lesso:n^  XL 

TENDON. 

Tendon  is  composed  of  white  fibres  arranged  longitudinally  and 
parallel  to  each  other.  The  fibres  are  arranged  in  bundles,  the 
tendon-bundles^  which  are  held  together  by  a sheath  and  septa  of 
connective-tissue.  The  fibres  are  united  to 
each  other  by  a cement  sid)stance,  and  on 
the  primary  bundles  of  the  fibres  are  placed 
the  tepdon-cells,  which  vary  in  their  shape 
and  arrangement  in  different  tendons.  It 
is  supplied  by  few  blood-vessels,  and  con- 
tains only  a few  elastic  fibres. 

Tendon. — Harden  a small  tendon  of  a 
man,  calf,  dog,  or  cat,  in  Miiller’s  fluid, 
alcohol,  bichromate  of  potash,  and  com- 
plete the  hardening  in  alcohol.  By  freez- 
ing, make  transverse  and  longitudinal  sec- 
tions, or  use  the  celloidin  method.  Tendons 
cannot  be  cut  after  being  embedded  in 
paraffin,  they  become  too  hard. 

1.  T.S.  Tendon  (L). — (a.)  Stain  a sec- 
tion in  picro-carmine,  and  mount  it  in 
Farrant’s  solution.  Observe  the  sheath 
(fig.  129,5),  composed  of  connective  tissue 
arranged  circularly,  sending  septa  (t)  into 
the  substance  of  the  tendon,  thus  breaking  it  up  into  polygonal 
areas  of  different  sizes,  which  are  filled  by  the  cut  encls  of  the 
longitudinally-arranged  fibres. 

^ Comptes  Eendus,  vol.  Ill,  p.  863,  1890. 


sel ; t.  Trabeciilse  or  septa  ; 
c.  Branched  spaces  in  the 
tendon  for  tendon-cells; 
1.  Matrix  or  cut  ends  of 
the  fibres,  x 50. 


164 


PRACTICAL  HISTOLOGY. 


[XL 


(h.)  The  brancliiiig  stellate  spaces,  interfascicular  spaces  (fig. 
129,  c),  between  the  fasciculi  or  bundles  of  fibres.  If  these  spaces 
contain  air,  they  appear  somewhat  dark.  These  spaces  can  readily 
be  seen  as  branched  dark  spaces  if  a transverse  section  is  made — 
by  means  of  a knife,  not  a razor — of  a small  tendon  dried  at  the 
ordinary  temperature. 

(c.)  (H)  Observe  the  cut  ends  of  the  fibres  (/),  which  appear 
almost  homogeneous,  but  amongst  them  here  and  there  may  be 
seen  a few  dots,  which  are  the  transverse  sections  of  elastic  fibres, 

S:Z  and  the  branched  tendon- 

spaces  (c),  some  of  them 
with  a nucleated  branched 
cell. 

2.  L.S.  Tendon. — Stain 
a section  with  logwood,  and 
mount  it  in  balsam.  (H) 
Observe  the  longitudinal 
arrangement  of  the  fibres^ 
and  between  them  rows  of 
fusiform  tendon -cells,  or 
rather  the  long  fusiform 
nuclei  of  the  tendon  cells 
arranged  between  the 
fibres.  The  other  parts  of 
the  cell  become  too  trans- 
])arent  to  be  seen  (fig.  130). 
Sometimes  a L.S.  of  one  of 
the  septa  may  be  seen. 

3.  Fibrils  in  Tendon 
(H).  — Macerate  a tendon 
from  the  tail  of  a rat  for 
twenty-four  hours  in  a satu- 
rated solution  of  picric  acid, 

3-4  hours  in  baryta 
Is.  water.  Tease  a small  part, 

and  examine  it  first  in 
water.  Mount  it  in  Farrant^s  solution.  This  is  apt,  however,  to 
render  the  fibrils  too  transparent.  Perhaps  a better  method  is  to 
place  the  fine  tendons  for  twenty-four  hours  in  equal  parts  of 
I per  cent,  osniic  acid  and  i per  cent,  silver  nitrate.  Mount  a 
teased  preparation  in  Tarrant’s  solution. 

{a.)  Observe  the  isolated  fibrils  inch  in  diameter),  exces- 

sively fine,  wavy,  and  unbranched  (fig.  131). 

4.  Tendon  of  Rat  [Gold  Chloride  Method). — Kill  a rat,  cut  off 
its  tail,  forcibly  rupture  the  tail,  when  a long  leash  of  fine  white 


XL] 


TENDON. 


165 


glistening  threads  or  tendons  will  be  obtained.  Prepare  the 
tendons  by  one  of  the  gold  chloride  methods.  One  of  the  best 
methods  is  the  lemon-juice  method  of  Panvier  (p.  79),  but  the 
boiled  formic  acid  and  gold  method  also 
yields  excellent  results.  It  is  to  be  re- 
membered that  it  is  not  necessary  to  use 
gold  chloride  to  demonstrate  the  tendon- 
cells  ; this  can  be  done  by  hsematoxylin. 

With  regard  to  the  action  of  gold 
chloride,  my  experience  leads  me  to 
believe,  that  in  order  to  see  the  rows  of 
tendon-cells  with  their  lateral  protoplasm 
expansions,  the  lemon-juice  method  is 
very  good ; while  the  old  acetic  acid 
method  makes  the  fibres  less  swollen  up, 
and  on  teasing  they  a;e  readily  isolated, 
thus  enabling  one  to  see  cells  either 
singly  or  in  rows  clasping  them.  Not  un- 
frequently  isolated  tendon-cells  are  to  be 
seen  in  the  field  of  the  microscope. 

Tease  a small  part  of  the  gold  tendon  in  FarranTs  solution. 

(a,)  (H)  The  fibres  arc  swollen  up  and  transparent,  and  lying  on 
them  are  rows  of  tendon-cells  (fig.  132,  h,  h)  stained  of  a violet 
tint.  ' Each  cell  h somewhat  oblong  with  a distinct  nucleus,  and 


Fia.  T31. — Fibrils  of  Tendon  of 
Rat  Isolated  by  Picric  Acid, 
X 300. 


Fig.  132. — Gold  Chloride, 
Tendon,  Tail  of  P^at.  a. 
Tendon  - cells  seen  on 
edge  and  embracing  a 
fibre;  bb.  On  the  flat, 
the  cells  with  a ridge. 


cell  protoplasm  ; n.  Nucleus ; a.  Stripe  or 
ridge. 


bears  a flattened  wing-shaped  expansion  (fig.  133).  Along  the  cells 
is  usually  to  be  seen  a stripe  or  ridge  (fig.  133),  produced  by  the 
cells  being  compressed  between  several  adjoining  fibres.  This  ridge 
may  be  seen  to  be  interrupted  in  some  of  the  cells.  The  nuclei  of 
the  adjacent  cells  may  be  seen  to  be  close  together. 


PRACTICAL  HISTOLOGY. 


[XL 


1 66 


(h.)  If  a,  side  view  of  the  cells  is  obtained  (fig*  132,  a),  they 
partially  clasp  the  fibre,  but  never  envelope  it  completely ; in  this 
respect  these  cells  differ  from  endothelium. 

5.  T.S.  of  Gold  Tendon  (H). — Remove  the  skin  from  the  tail  of 
a young  rat,  cut  out  a piece  of  the  tail  a quarter  of  an  inch  in 
length  with  its  tendons,  and  subject  it  to  the  gold  chloride  process 
(p.  79).  When,  after  reduction,  it  has  become  purple  or  brownish, 
decalcify  the  bone,  harden  it  in  alcohol,  and  make  transverse 
sections.  Mount  one  in  balsam. 

(a.)  Many  tissues  will  be  seen,  including  muscle,  nerve,  fat,  and 
bone.  hTeglecting  these,  observe  the  small  rounded  areas  at  the 
circumference,  the  transverse  sections  of  the  small  tendons,  each 
surrounded  by  its  own  sheath  of  connective  tissue  (fig.  134,  t).  In 
each  observe  the  branched  stellate  spaces  (fig.  134,  c),  frequently 
anastomosing  with  each  other.  These  interfascicular  spaces  are 


Fig.  134.— T.S.  of  a small  Ten- 
don, Tail  of  Rat.  t.  Sheath ; 
c.  Interfascicular  spaces 
with  tendon -cells.  Gold 
chloride. 


Fig.  135. — Layer  of  Endo- 
thelial Cells  on  the  Sur- 
face of  a Tendon,  Tail  of 
Rat.  Silver  nitrate. 


purplish  in  colour ; they  contain  the  tendon-cells,  and  also  a 
purplish  deposit  due  to  the  gold  chloride  acting  on  the  lymph  which 
they  contain  in  the  fresh  condition. 

6.  Endothelial  Sheath  of  Tendon  (L  and  H). — Silver  a leash  of 
the  fine  tendons  from  the  tail  of  a rat.  Mount  a short  length  of 
one  of  them  in  balsam. 

(a.)  Observe  the  tendon  made  u]>  of  parallel  fibres,  and  note  on 
their  surface  a single  layer  of  endothelium.  The  squames  are  large, 
polygonal,  and  mapped  out  by  “silver  lines,”  but  no  nuclei  are 
visilfie. 

7.  Fresh  Tendons  and  Acid  Logwood  (H). — Place  three  or  four 
tendons  (rat’s)  inch  long,  on  a d)y  slide,  and  fix  their  ends  with 
paraffin,  so  as  to  keep  them  extended.  Make  a solution  of  acid 
logwood  by  adding  one  part  of  1 per  cent,  glacial  acetic  acid  to 
three  parts  of  logwood  solution.  This  solution  is  red.  Place  a 
drop  of  it  on  a cover-glass,  and  lay  it  on  the  tendons.  The  acid 
brings  into  view  rows  of  narrow,  granular,  nucleated  cells  between 


XL] 


TENDON. 


167 

the  fibres  of  the  tendon,  while  at  the  same  time  the  logwood  stains 
them.  Instead  of  acid  logwood  use  picro-carmine  or  acid  hsema- 
toxylin  (p.  69).  Displace  the  dye  with  water  and  mount  in 
glycerine.  The  tendons  are  purposely  taken  longer  than  the 
breadth  of  the  cover-glass,  so  that  they  may  remain  stretched. 

8.  Fresh  Tendon. ^ — On  a black  surface,  tease  in  normal  saline  a 
small  piece  of  any  tendon  of  a calf.  Observe  the  fibres  and  fibrils, 
but  no  cells  are  visible.  Irrigate  the  preparation  with  2 per  cent, 
acetic  acid.  The  mass  becomes  clear  and  transparent  to  the  naked 
eye,  and  now  under  the  microscope  one  sees  the  fusiform  nucleated 
cells  singly  or  in  line  in  order  between  the  swollen-up  fibres.  The 
preparation  may  be  stained  with  magenta,  which  brings  into  clearer 
view  the  cells,  and  any  elastic  fibres  present, 


ADDITIONAL  EXERCISES. 


9.  Tendon  of  Rat  {DogieVs  Method). — Very  good  preparations  are  obtained 
by  placing  the  fresh  tendons  for  several  days — tlie  longer  the  better— in 
Grenadiers  alum -carmine  (p.  65).  This  fluid  stains  but  slowly.  The  cells, 
however,  are  stained,  and  if  a tendon  be  teased,  isolated  cells,  and  cells  on  the 
fibres  are  easily  seen.  It  is  a good  method  for  showing  the  relations  of  the 
cells  to  the  fibres. 

10.  -T.S.  Tail  of  Rat  {Corrosive  Sublimate  and  Borax-Carmine). — Harden 
short  lengths  of  the  tail  of  a rat,  the  skin  being  first  removed,  in  corrosive 
sublimate  for  three  hours  or  so.  Remove  every  trace  of  the  mercuric  salt 
by  prolonged  washing  in  alcohol.  Stain  the  tissue  in  bulk  in  borax-car- 
mine, and  then  decalcify  it  in  dilute  hydrochloric  acid.  Make  transverse 
sections,  after  embedding  it  by  the  interstitial  method  in  paraffin.  Sections 
may  also  be  made  by  freezing,  but  they  are  apt 

to  fall  asunder.  This  method  also  yields 
beautiful  preparations,  comparable  to  those  by 
the  gold  chloride  methods.  The  transverse 
sections  of  the  tendons  are  very  characteristic. 

11.  Dried  Rat’s  Tendons. — A very  convenient 
method  is  to  dry  the  tendons  of  a rat’s  tail, 
keeping  them  extended  during  the  process. 

After  drying,  they  can  then  be  used  at  any 
time.  By  acting  on  them  with  dilute  acetic 
acid  they  swell  up  slowlv,  and  the  rows  of  cells 
are  thereby  revealed.  The  cells — after  washing 
away  the  acetic  acid— can  be  stained  with  picro- 
carmine,  and  the  preparation  mounted  in  dilute 
glycerine. 

12.  Cell-Spaces  {Saft-Candlchen)  in  Central 
Tendon  (Silver  Method). —VIslcq-  the  central 
tendon  or  the  whole  diaphragm  for  five  minutes 
in  a J per  cent,  silver  nitrate.  Remove  it,  and 
with  a camel’s-hair  pencil  brush  both  surfaces 

of  the  tendon  to  remove  the  endothelium.  Replace  it  in  the  silver  solution 
for  fifteen  minutes.  Remove  it ; wash  it  in  water,  and  expose  it  to  light  to 


Fig.  136.— Cell-Spaces  in  the  Cen- 
tral Tendon  of  the  Diaphragm 
of  Rabbit.  I Lymphatic  ; s. 
Cell-spaces.  Silver  nitrate. 


PRACTICAL  HISTOLOGY. 


[XII. 


1 68 

reduce  the  silver.  One  piece  maybe  mounted  in  balsam  ; another  piece  should 
be  stained  with  ncid  logwood  or  picro-lithium  carmine  (in  this  case  use  dilute 
hydrochloric  acid),  anil  mounted  in  balsam. 

(a.)  Observe  a large  number  of  clear,  branched  anastomosing  spaces,  sur- 
rounded by  brown  areas  of  ground-substance.  The  former  are  the  cell-spaces 
and  Saft-Candlchcn^  or  juice-canals,  and  some  of  the  latter  may  be  seen  to 
communicate  with  the  lymphatics  (fig.  136). 

{h.)  In  the  stained  specimen,  stained  nuclei  are  seen  in  the  spaces,  , the 
nuclei  of  the  cells  which  occupy  these  spaces. 

13.  Cell-Spaces  {Iron  Sulphate  Method). — Using  the  fresh  central  tendon  of 
the  diaphragm  of  a mouse  or  guinea-pig  or  rat,  place  it  for  a few  minutes  in  i per 
cent,  sulpliate  of  iron.  Pencil  away  the  surface  endothelium,  and  leave  it  in 
the  iron  solution  for  five  to  seven  minutes.  Remove  it,  wash  it,  and  place  it 
in  I per  cent,  ferricyanide  of  potash,  in  which  it  becomes  blue.  Mount  it  in 
Tarrant’s  solution  or  balsam.  In  this  preparation  the  cell-spaces  and  juice- 
canals  are  again  clear,  but  the  ground-substance  is  blue. 

14.  Cell-Spaces  in  Rat’s  Tendon. — The  fine  tendons  are  placed  in  silver 
nitrate  (J  per  cent.)  for  two  minutes,  and  then  the  epithelium  is  brushed  off 
by  a camel’s-hair  pencil.  Five  or  six  sweeps  of  the  brush  usually  suffice.  The 
tendons  are  stained  for  other  ten  minutes  in  silver,  washed,  and  exposed  to 
light  in  alcohol.  Rows  of  clear,  somewhat  quadrangular  spaces  in  a brownish 
matrix  are  obtained. 


LESSON  XII. 

ADIPOSE,  MUCOUS,  AND  ADENOID  TISSUES— 
PIQMENT  CELLS. 

ADIPOSE  TISSUE  (FATTY  TISSUE). 

Adipose  Tissue. — A fat-cell  consists  of  a membrane  enclosing  a 
globule  of  oil^  which  pushes  the  oval  flattened  nucleus  (surrounded 
by  a small  amount  of  protoplasm)  to  one  side,  so  that  it  lies  close 
under  the  cell-wall.  Size,  40  /x  to  80  /x  (^^^-3^0  i^^ch). 

Fat-cells  are  arranged  in  groups,  which  form  lobules,  and  these 
again  form  lobes.  Each  lobule  has  an  afferent  artery,  one  or  two 
efferent  veins,  and  a dense  network  of  capillaries  between  the  fat- 
cells,  each  capillary  surrounding  one  or  more  fat-cells. 

It  is  to  be  remembered  that  cells  in  connective  tissue  containing 
fat  may  have  a two-fold  origin.  Eat  may  be  formed  in  ordinary 
connective-tissue  cells,  but  there  are  other  cells  of  a connective- 
tissue  nature,  which  seem  to  be  more  specifically  fat-cells.  During 
development  this  tissue  is  formed  at  certain  parts,  e g.,  in  the  groin, 
axilla,  and  neck,  and  presents  a grayish-yellow  appearance  in  the 
form  of  lobules,  surrounded  by  connective-tissue — readily  seen 
in  a young  animal.  The  cells  at  first  contain  granules.  The 


ADIPOSE  TISSUE. 


XII.] 


169 


Fig.  137. — Fat-Cells,  some 
showing  a Nucleus. 
The  central  one  shows 
crystals  of  margarine, 
X 100. 


fat-cells  derived  from  this  tissue  form  lobules,  and  arc  su})plied 
with  blood-vessels  very  much  as  a gland  is 
supplied  with  blood ; so  that  each  lobule  is 
jirovided  with  an  artery,  vein,  and  capillaries. 

1.  Fat-Cells. — Cut  out  a small  piece  of  the 
omentum  of  a cat,  selecting  a piece  that  con- 
tains a little  fat,  and  mount  it  in  normal  saline. 

(a.)  (L)  Observe  the  large,  highly  refractive 
fat-cells  arranged  singly  or  in  groups  (fig.  137). 

(b.)  (H)  The  large  fat-cells  of  variable  size 
— some  of  them  2)olygonal — highly  refractive 
contents,  but  no  nucleus  visible ; connective 
tissue  passing  between  some  of  the  cells. 

2.  Action  of  Osmic  Acid  (H). — Place  a small  part  of  the  omen- 
tum of  ft  cat  or  embryo 
ox  in  ^ per  cent,  osmic 
acid  for  an  hour.  Wash 
it  thoroughly,  and  mount 
in  Farrant’s  solution. 

(a.)  (L)  Observe  the 
fat-cells,  which  first  be- 
come, brown  and  ulti- 
mately quite  black.  The 
fat-cells  are  in  groups, 
and  their  relations  to  the 
blood-vessels  can  also  be 
seen  in  fig.  138. 

3.  Membrane  and 
Nucleus  of  Fat-Cells 
(H).  — Place  a piece  of 
the  omentum  or  subcu- 
taneous adipose  tissue 
in  absolute  alcohol  for 
several  days,  and  after- 
wards in  ether  for  a day 
or  two ; or  the  tissue 
may  be  boiled  for  a few 
minutes  first  in  alcohol 
and  then  in  ether. 

Transfer  a small  piece 
to  haematoxylin  and 
allow  it  to  stain  for 
several  hours.  Wash  it 
in  water  and  place  it  in 

absolute  alcohol.  Extract  it  with  turpentine,  clear  it  up  with  clove- 
oil,  and  mount  it  in  balsam. 

16 


B 


Fig.  138. — Fat-Cells  stained  w ith  Osmic  Acid  from  thi 
Omentum  of  an  Embryo  Ox.  A.  The  fat-cells  in 
groups  or  lobules,  blackened  by  the  osmic  acid,  and 
showing  their  relation  to  the  blood-vessels ; JD.  a-e 
fat-cells  in  different  stages  of  development. 


170 


PRACTICAL  HISTOLOGY. 


[XII. 


(a,)  Observe  the  collapsed  membrane  of  the  fat-cells,  with  a small 
oval  blue-stained  nucleus  immediately  under  the  cell-wall.  A small 
quantity  of  protoplasm  surrounding  the  nucleus  may  be  visible. 

4.  Or  harden  a small  piece  of  the  omentum  of  a rat  or  other 
animal  in  absolute  alcohol.  Select  a piece  which  contains  some  fat. 
Stain  it  for  twenty  minutes  in  lithium-carmine,  then  place  it  in  acid 
alcohol  (i  per  cent,  hydrochloric  acid  in  70  per  cent,  alcohol)  ; 
place  it  in  absolute  alcohol ; clear  it  up  with  clove-oil  or  xylol,  and 
mount  in  Canada  balsam. 

(a.)  Observe  the  envelopes  of  the  fat-cells  and  the  nuclei  of  the 
fat-cells,  the  latter  stained  bright  red  (fig.  139).  Other  red-stained 
nuclei  are  visible,  but  they  are  the  nuclei  of  blood-capillaries  between 
the  fat-cells. 

5.  Margarine  Crystals  (H). — Place  a small  piece  of  fat  for  forty- 
eight  hours  in  glycerine.  Tease  a piece  in  Farrant’s  solution. 

(a.^  FTotice  the  large  cells  some  of  them  with  granular  contents, 


Fig.  140.— Fat-Cells  containing  Crystals  of 
Margarine. 


others  with  a stellate  arrangement  of  needle-shaped  crystals  of 
margarine  (figs.  137  and  1 40).  If  the  star  of  cr^^stals — really  palmitic 
and  stearic  acids — be  broken  up,  then  the  needle-shaped  crystals  are 
distributed  throughout  the  cell.  ^ 

6.  Blood-Vessels  of  Adipose  Tissue  (L  and  H). — Make  a rather 
thick  section  of  a mass  of  adipose  tissue  in  which  the  blood-vessels 
have  been  injected.  This  can  be  done  by  injecting  the  blood-vessels 
of  a rabbit  with  a carmine-gelatine  mass.  As  the  fat  is  very  soft,  the 
best  method  of  obtaining  such  sections  is  to  saturate  the  tissue  with 
paraffin  and  cut  it  in  paraffin.  Mount  sections  — not  too  thin — in 
balsam.  A section  which  has  been  saturated  with  paraffin  and  cut 
in  this  substance  must  have  the  paraffin  removed  by  soaking  in 


ADIPOSE  TISSUE. 


XII.] 


171 


turpentine  or  xylol,  which  dissolves  out  the  paraffin  ; the  section  is 
afterwards  mounted  in  balsam. 

(L)  Observe  the  very  vascular  small  lobules^  composed  of  fat-cells. 
To  each  lobule  there  passes  one  artery,  and  from  it  emerge  one  or 
two  veins. 

(H)  Observe  the  loop  of  capillaries  round  each  fat-cell  or  around 
several  fat-cells. 

7.  Development  of  Fat-Cells. — These  may  he  studied  in  the 
subcutaneous  tissue  of  a newly-born  rat,  or  in  the  omentum  of  a 
newly-born  rabbit.  Stain  a small 
piece  of  any  of  these  tissues  in  osmic 
acid. 

(a.)  (L)  Observe  the  fat-cells  in 
groups  surrounded  by  connective 
tissue. 

{h.)  (H)  Observe  the  shape  of  the 
cells,  with  small  globules  of  oil — 
black  — scattered  throughout  the 
protoplasm,  the  nucleus  pushed  to 
one  side  in  the  more  developed  fatty 
cells  by  a globule  of  oil,  which  is 
stained  black  (figs.  138,  141). 

8.  Atrophic  Fat-Cells. — These  are 
readily  obtained  from  the  yellow 
hone-marrow  of  an  old  person  who  has  died  from  some  wasting 
disease,  or  from  the  sub-pericardial  fat  of  a person  who  has  died 
from  phthisis. 

(H)  Observe  the  envelope  of  the  fat-cell,  now  no  longer  completely 
filled  with  fat,  but  containing  a little  protoplasm  and  some  serous 
fluid. 

9.  Injection  Method. — By  means  of  a hypodermic  syringe,  inject 
under  the  skin  of  a dog  or  cat  silver  nitrate  (i  in  1000),  which 
causes  a local  oedema  and  isolates  the  elements  of  the  areolar  tissue 
including  the  fat-cells  {Ranvier), 


Fig.  141.— Developing  Fat-Cells  from 
the  Subcutaneous  Tissue  of  a 
Foetus.  Osmic  acid. 


MUCOUS  TISSUE. 

Mucous  Tissue. — In-  the  embryo  it  exists  under  the  skin  ; it 
forms  Wharton's  jelly  of  the  umbilical  cord,  and  in  the  adult  it  forms 
the  vitreous  humour.  It  is  essentially  an  embryonic  tissue. 

10.  Mucous  Tissue  (H). — Harden  the  umbilical  cord  of  a three- 
months  foetus  in  Muller's  fluid  and  then  in  alcohol.  Make  trans- 
verse sections  by  freezing,  and  stain  them  in  picro-carmine  or 
hsematoxylin  or  gentian- violet.  Mount  in  Farrant's  solution. 

(a.)  Observe  the  large,  branched,  granular,  nucleated  cells,  which 


1/2 


PRACTICAL  HISTOLOGY. 


[XIL 


anastomose  with  each  other.  Between  the  cells  is  a fluid  which 
contains  mucin,  and  according  to  the  stage  of  development  of  the  cord, 
there  is  a greater  or  less  number  of  fibres.  The  older  the  cord,  the 

more  the  fibres  increase  in  num- 
ber, and  its  characters  approach 
those  of  ordinary  connective- 
tissue.  A better  view  of  the 
finer  processes  is  obtained  by 
examining  the  tissue  in  normal 
saline  (fig.  142). 


Fig.  142. — Mucous  Tissue  of  Umbilical  Cord 
of  Foetus.  Muller’s  fluid  and  logwood,  x 
300. 


ADENOID  TISSUE, 

Adenoid,  Retiform,  or  Re- 
ticular Tissue  consists  of  a re- 
ticulum or  network  of  fine  fibrils, 
which  run  in  all  directions, 
forming  a meshwork  in  several 
planes.  Some  regard  it  as  made 
up  of  branched  corpuscles,  the  processes  of  which  anastomose. 
In  the  meshes  are  leucocytes  or  lymph-cells,  which  usually  occur 
in  such  numbers  as  to  obscure  the  presence  of  the  fine  meshwork 
in  which  they  lie. 

It  is  very  widely  distributed,  ejj.,  in  lymphatic  glands,  simple 
and  compound,  tonsils,  solitary  glands,  and  Peyer’s  glands ; in  the 
bronchial,  pharyngeal,  nasal,  intestinal  mucous  membrane,  spleen, 
thymus,  and  a few  other  situations. 

11.  Adenoid  Tissue  of  Lymphatic  Glands. — This  may  be  pre- 
pared in  several  ways. 

(i.)  Harden  an  abdominal  lymphatic  gland  of  a calf  or  kitten  for 
two  weeks  in  Muller^s  fluid.  Make  sections,  and  shake  up  one  in  a 
test-tube  with  some  water  ; this  dislodges  the  lymph  corpuscles,  and 
in  places  leaves  the  fine  reticulum  visible. 

(ii.)  A better  plan  is  to  inject  into  a fresh  lymph  gland  a ^ per 
cent,  solution  of  osmic  acid,  or  \ per  cent,  solution  of  silver  nitrate. 
In  either  case  an  oedema  is  produced  which  separates  the  parts  and 
reveals  the  network.  The  injection  is  made  by  means  of  a hypo- 
dermic syringe  (fig.  1 26).  The  syringe  is  filled  with  the  solution,  and 
the  sharp  nozzle  is  thrust  into  the  gland,  and  the  contents  of  the 
syringe  rapidly  injected  haphazard  into  the  organ.  It  passes  in,  and 
forms,  as  it  were,  an  oedema,  and  separates,  and  at  the  same  time 
hardens,  the  constituent  parts  of  the  organ.  This  is  the  method  of 
interstitial  injection,  one  which  is  frequently  employed. 

(iii.)  The  gland  may  be  hardened  for  twenty-four  hours  in  picric 


ADENOID  TISSUE. 


XII.] 


173 


acid,  and  the  sections  stained  with  eosin-hsematoxylin  and  mounted 
in  balsam. 

(H)  Observe  some  parts  crowded  with  lymph  corpuscles,  but  where 
these  are  wanting,  note  the  very  fine  network  of  fibres,  with  nuclei 
here  and  there  at  the  points  of  intersection  (fig.  143). 

The  lymph  corpuscles  may  be  got  rid  of  by  applying  to  a fresh 
preparation  a dilute  solution  of  caustic  potash  which  dissolves  them. 

12.  Pigment-Cells  and  Guanin-Cells. — These  may  be  studied 
by  pinning  out  on  a frog-board  one  of  the  webs  between  the  toes  of 
a frog  (Lesson  XIX.  11,  e). 

(a.)  (Land  H)  Observe  large,  branched,  corpuscles  loaded  with 


Fig.  143- — Adeiiuid  Tissue.  Human  lymphatic 
gland.  Picric  acid  and  eosin-haematoxylin. 


Fig.  144.— Pigment  and  Guanin-Cells  of 
Frog.  A.  Contracted  ; B,  C.  Partially 
relaxed  pigment-cells.  G.  Guanin 
cells. 


black  granules  of  melanin  (fig.  144,  B),  also  smaller  black  spots, 
which  are  cells  with  their  processes  retracted.  Every  intermediate 
stage  between  these  two  states  may  be  seen. 

A permanent  preparation  is  readily  made  by  stripping  off  the  skin 
from  the  web  of  the  toe  of  a dead  frog,  hardening  it  in  alcohol,  and 
mounting  in  balsam.  The  web  should  be  fixed  in  an  extended 
position  before  it  is  placed  in  the  alcohol. 

In  such  a preparation,  not  only  will  };igment-cells  be  found,  but 
guanin-cells  also,  i.e.^  small  oval  cells  filled  with  white  refractive 
granules  of  guanin  (fig.  144,  G).  To  see  the  guanin-cells  turn  off 
the  light  reflection  from  the  mirror,  when  the  granules  in  the  guanin- 
cells  will  appear  bright  and  retractile  on  a black  ground. 

For  pigmented  connective-tissue  corpuscles  from  the  choroid,  see 
Lesson  on  Eye. 


174 


PRACTICAL  HISTOLOGY. 


[XIII. 


ADDITIONAL  EXERCISE. 

13.  Mucous  Tissue. — Many  branched  cells  are  seen  in  a T.S.  of  the  tail  of  a 
tadpole,  young  triton,  or  salamander,  hardened  in  i per  cent,  osmic  acid  and 
cut  in  paraffin. 

It  is  also  to  be  found  under  the  skin  of  the  flanks  in  frogs  at  the  breeding 
season.  It  gives  good  preparations  when  stained  with  methyl-violet-5 B 
{S.  Mayer). 


LESSON  XIII. 

BONE,  OSSEOUS  TISSUE,  &c. 

The  essential  elements  of  osseous  tissue,  of  which  bone  consists, 
are  a calcified  filjrous  matrix  or  ground-substance,  with  cells  or 
hone-corpuscles  embedded  in  it ; the  latter  are  lodged  in  spaces 
called  lacunoe.  A bone,  however,  is  a complex  organ.  The  follow- 
ing scheme  may  facilitate  the  comprehension  of  its  minute 
structure : — 

BONE. 

In  a longitudinal  section  of  a long  bone,  observe  with  the  naked 
eye— 

Periosteum  covering  the  bone. 

Compact  or  dense  bone,  the  substantia  dura  (with  Haversian 
canals). 

Cancellated  or  spongy  bone,  the  substantia  spongiosa  (with 
Haversian  spaces  and  cancelli). 

Medullary  cavity  with  marrow. 

Histologically  dry  compact  bone  shows — 

[Peripheric  or  circumferential. 
j I Haversian  or  concentric. 

ame  ce-  interstitial  or  ground. 

[Perimedullary. 

Sharpey's  perforating  fibres  | 

Haversian  System. 

Recent  hone. 

Blood-vessels,  connective  tissue, 
lymphatics,  osteoblasts. 
Between  the  lamellae,  branched 
bone-corpuscles. 

Processes  of  bone-corpuscles 
and  lymph. 

Lymph. 


Dry  hone. 

Haversian  canal  . 

„ lamellae  . 

Lacunae  . . . . 

Canaliculi . . . . 


XIII.] 


BONE,  OSSEOUS  TISSUE,  ETC.  1/5 

f I.  External  layer,  fibrous,  with  the  larger  blood- 
j vessels. 

Periosteum  \ 2.  Internal  or  osteogenic  layer,  with  finer  hlood- 
j vessels,  numerous  elastic  fibres,  and  osteo- 

I blasts^  and  sometimes  osteoclasts. 

Osteoclasts  or  myeloplaxes  of  llobin. 

Blood-vessels,  nerves,  and  lymphatics. 

f I.  Endochondral  in  cartilage. 

Development  j Intra-membranous  or  periosteal. 


1.  T.S.  of  Dense  or  Compact 
Bone. — It  is  better  to  buy  a pre- 
pared transverse  section  of  the 
shaft  of  a small,  dry,  long  bone. 
In  it  most  of  the  smaller  spaces, 
being  filled  with  air,  appear 
black. 

{a.)  (L)  Observe  the  medullary 
cavity,  bounded  by  a ring  of  bone, 
less  dense  internally. 

(6.)  Sections  of  tubes  which 
appear  round,  or  oval — Haver- 
sian canals — surrounded  by  con- 
centric lamellae  (fig.  145,  c),  and 
between  these  lamellae  are  lacunae 
(e),  with  fine  channels  passing 
from  them — the  canalicuK.  Some 
lamellae  are  arranged  parallel  to 
the  circumference  of  the  bone — 
the  peripheric  (a) — while  others 
of  larger  radius  are  incomplete, 
and  jammed  in  between  the 
Haversian  system : they  are  inter- 
mediary (d).  Around  the  medul- 
lary canal  are  the  perimedullary 
lamellae  {h). 

(c.)  (H)  Observe  the  shape  of 
the  lacunae — flattened  branched 
spaces  — with  their  numerous 
^ wavy  branching  canaliculi,  and 
how  adjoining  canaliculi  anasto- 
mose by  traversing  the  lamellae 
(figs.  145,  147,  e).  At  the  outer 
part  of  each  Haversian  system, 
some  of  the  canaliculi  of  the 


Fig.  145. — T.S.  Human  Metacarpal  Bone. 
a.  Peripheric  lamellae ; b.  Perimedullary 
lamellae ; c.  Haversian  canals  sur- 
rounded by  their  Haversian  lamellae  ; 
d.  Intermediary  lamellae;  e.  Lacunae, 
X 20. 


176  PRACTICAL  HISTOLOGY.  [XIII 

outermost  row  of  lacunse  will  be  found  to  form  loops  and  open  into 
the  lacunm  from  which  they  arose : these  arc  recurrent  canaliculi 
(fig.  147,  a).  The  canalicular  system  is  for  the  listribution  of 
lymph  to  all  the  parts  of  the  calcified  fibrous  matrix.  iN’otice  also 
that  the  intermediary  lamellm  are  parts  of  circles  with  a much 

larger'  radius  than  those 
of  the  Haversian  system. 
Each  Haversian  canal, 
with  its  system  of  lamel- 
lae, lacunae,  and  canali- 
culi, forms  a Haversian 
System.  The  greatest 
diameter  of  the  lacunae  is 
about  14  fx  (ygVo  iiieh). 

(d,)  The  lamellae  in  a 
Haversian  system  on 
transverse  section  appear 
as  thin  concentric  bands, 
a clear  transparent  one 
alternating  with  one  which 
looks  more  granular. 
These  are  not  due  to 
different  kinds  of  lamellae, 
but  in  the  clear  ones  one 
looks  upon  the  long  axis 
of  the  fibrils  composing  the  lamella,  and  in  the  others  upon  the 
ends  of  the  fibrils. 

2.  L.S,  of  Dense  Bone,  prepared  in  the  same  way. 

(a.)  (L)  Observe  Haversian  canals  running  chiefly  in  the  long 
axis  of  the  section,  with  here  and  there  oblique,  short,  junction 
canals.  Hear  the  surfaces  some  open  externally,  and  others  com- 
municate with  the  medullary  cavity.  The  lamellae  of  any  Haver- 
sian system  run  parallel  to  its  own  Haversian  canal.  In  the 
system  of  canals — each  20-100  /x  wide — the  canals  frequently 
divide  dichotomously,  and  ultimately  form  a network  in  the  com- 
pact bony  substance. 

(b.)  If  the  section  be  near  the  surface  of  the  bone,  so  as  to 
include  the  peripheric  lamellae,  canals  for  blood-vessels,  perforating 
the  lamellae  and  not  surrounded  by  lamellae  as  in  the  Haversian 
systems,  may  be  seen.  They  are  called  Volkmann’s  canals,  and 
contain  the  perforating  vessels.  They  are  connected  with  the 
Haversian  canals  ])roper.  They  are  well  seen  in  sections  of  the 
femur  of  a guinea-pig,  but,  unlike  Haversian  canals,  they  are  not 
surrounded  by  Haversian  or  concentric  lamellae. 

(c,)  (H)  Observe  the  flattened  oval  lacunae  with  their  canaliculi; 


Fig.  146. — T.S.  The  Shaft  of  a Human  Femur.  II. 
Haversian  canals;  s.  Haversian  lamella?;  si.  In- 
termediary lamellae,  x 40. 


XIII.] 


BONE,  OSSEOUS  TISSUE,  ETC. 


177 


their  arrangement,  as  well  as  that  of  the  lamellae  (fig.  148).  If  a 
canal  he  viewed  carefully  many  fine  dots  will  be  seen  in  it.  These 
are  the  openings  of  the  canaliculi. 

3.  T.S.  Decalcified  Shaft  of  a Bone. — The  hone,  cut  into  short 
lengths,  must  he  decalcified  in  picric  acid  or  chromic  and  nitric 
fluid,  with  the  precautions  laid  down  at  p.  38.  When  sufliciently 


c 


Fig.  147.— T.S.  Shaft  of  Human  Femur.  II.  Haversian 
canals ; c.  Lacuiue  with  hone-corpuscles ; a.  Lacuna; 
with  recurrent  canaliculi ; s.  Intermediary  lamella;  with 
Sharpey’s  fibres,  h ; p.  Large  fibres  of  Sharpey  in  inter- 
mediary lamella; ; 1.  Confluent  lacuna;.  These  Ranvier 
supposes  are  bone-corpuscles  and  lacuna;  undergoing 
atrophy,  x 300. 


Fig.  148. — L.S.  Dense 
dry  bone,  x 40. 


soft — ascertained  by  pricking  it  with  a pin — it  is  hardened  in 
alcohol  in  the  usual  way.  Sections  are  best  made  by  freezing. 

(i.)  Stain  a section  in  picro-carmine  and  mount  it  in  glycerine- 
jelly.  Glycerine  or  Tarrant’s  solution  tends  to  make  the  prepara- 
tion rather  too  transparent. 

(ii.)  Place  some  sections  in  i per  cent,  osmic  acid  for  twenty-four 
hours  and  mount  them  in  glycerine-jelly. 

(a.)  (L)  Observe  the  periosteum  (fig.  149),  embracing  and 
adherent  to  the  bone.  In  the  bone  itself  the  lacunae,  and  especially 
the  canaliculi,  are  no  longer  black,  and  are  not  so  visible  as  in  the 
non-decalcified  bone.  Each  lacuna  contains  a highly  refractive, 
branched,  nucleated,  and  stained  corpuscle. 

Bone-Corpuscles. — Observe  their  arrangement  following  that  of 
17  M 


178 


PRACTICAL  HISTOLOGY. 


[XIII. 

the  lamellae,  but  the  latter  are  not  so  distinct  as  in  dry  bone. 
Several  lamellae  lie  between  two  consecutive  rows  of  bone-corpuscles. 
The  Haversian  canals  contain  blood-vessels,  connective  tissue,  and 
other  cells,  or  osteoblasts. 

(b.)  (H)  The  periosteum  consists  of  an  external  layer  stained 
red,  and  composed  chiefly  of  white  fibrous  tissue.  Attacdied  to  it 
may  be  found  small  fragments  of  striped  muscle.  The  internal 
layer  contains  many  elastic  fibres,  and,  especially  in  young  bones, 
there  may  be  seen  one  layer  or  more  of  flattened  or  cubical  cells, 


Pig.  149. — T.S.  Part  of  a Human  Metacarpal  Bone,  x 50.  h.  Haversian  space  with 
marrow.  Dilute  nitric  acid. 

called  osteoblasts  (fig.  156,  c).  The  latter  may  be  seen  not  only 
under  the  periosteum,  but  also  passing  along  with  blood-vessels  into 
the  Haversian  canals. 

Fibres  may  be  seen  passing  from  the  deep  surface  of  the  perios- 
teum into  the  bone — the  perforating  fibres  of  Sharpey. 

(c.)  Observe  the  lamellae,  but  their  outline  is  not  very  distinct, 
while  the  canaliculi  will  not  be  distinctly  visible,  being  indicated 
by  fine  lines  traversing  the  lamellae.  The  bone  corpuscles  are 
nucleated  refractive  cells,  each  lying  in  a lacuna.  In  such  a 
preparation,  one  cannot  make  out  that  they  send  processes  into  the 
canaliculi. 

(d.)  Select  a large  Haversian  canal  and  study  its  contents.  Note 
the  presence  of  an  artery  and  vein  with  very  thin  walls,  and  the 
cavity  lined  by  osteoblasts  (fig.  150,  Obi.),  and  the  remainder  filled 
up  with  medullary  tissue. 

4.  Perforating  Fibres. — (i.)  From  a membrane  bone  of  the 
skull  (e./7.,  the  parietal  or  frontal  bone,  which  has  been  softened 
in  2 per  cent,  hydrochloric  acid  or  in  v.  EbneFs  fluid  (p.  37) 


xm.] 


BONE,  OSSEOUS  TISSUE,  ETC. 


179 


Fig.  150. — T.S.  of  a large  Haversian  Canal  with  its 
Soft  Parts.  Femur  of  a D<ig,  x 130.  A.  Artery  ; 
V.  Vein  ; Kz.  Bone-cells  ; M.  Medullary  tissue  ; 
Obi.  Osteoblast ; Okl.  Osteoclast. 


and  from  which  the  acid  has  been  removed  by  steeping  in  water, 
and  subsequently  in  spirit)  with  forceps  remove  the  periosteum,  and 
tear  off  a thin  lamella  of 
osseous  tissue.  Place  its 
under-surface  uppermost  on 
a slide  in  water  or  v. 

Ebner’s  fluid. 

(H)  Observe  fine  tapering 
fibres  like  nails — perforat- 
ing fibres — projecting  from 
the  surface.  Some  aper- 
tures may  be  found  from 
which  corresponding  fibres 
have  been  withdrawn. 

(ii.)  These  are  far  better 
developed  in  the  bones  of 
the  skull  of  birds.  Soften 
the  vault  of  the  skull  of  a 

fowl  in  V.  Ebner’s  fluid  and  harden  in  alcohol.  Make  not  too  thin 
sections,  and  with  needles  tear  the  lamellae  asunder.  Examine  it 
in  V.  Ebner’s  fluid.  Or  make  sections  of  a human  frontal  bone 
softened  in  dilute  hydrochloric  acid,  and  examine  it  in  water. 

(H)  ^fumerous  perforating  fibres  passing  between  the  separated 
lamellae,  and,  it  may  be,  the  sockets  from  which  they  have  been 
withdrawn,  will  be  seen  (fig. 

1 5 1 ).  In  some  of  the  sections 
branched  perforating  fibres  are 
visible.  The  important  point 
is  to  make  the  sections  as 
nearly  as  possible  parallel  to 
the  course  of  the  fibres.  The 
preparation  is  apt  to  be  made 
too  transparent  by  Farrant’s 
solution,  so  that  the  fibres  are 
not  so  distinctly  seen  in  this 
medium  as  in  water  or  v. 

Ebner’s  fluid.  Observe  that  there  are  no  Sharpey’s  fibres  in  the 
Haversian  systems. 

5.  Blood-Vessels  of  Bone  (L). — These  are  not  easily  injected. 
Inject  with  a fluid  carmine  mass  (p.  89)  the  posterior  half  of 
the  body  of  a rabbit.  Do  this  from  the  abdominal  aorta.  Or  use 
the  injection  fluid  mentioned  at  p.  181.  It  requires  considerable 
pressure  to  cause  the  injection  to  traverse  the  blood-vessels  of  bone. 
Therefore  clamp  the  inferior  vena  cava  to  prevent  the  exit  of  the 
injection  mass.  Decalcify  the  injected  bone,  and  afterwards  make 


Fig.  151.— Sharpey’s  Perforating  Fibres. 


PRACTICAL  HISTOLOGY. 


i8o 


[XIII. 


T.S.  and  L.S.  The  sections,  however,  must  not  ho  too  thin.  L.S. 
are  the  most  instructive,  and  are  mounted  in  glycerine-jelly. 

(a.)  Observe  that  the  blood- 
vessels lie  in  the  Haversian 
canals,  and  follow  the  arrange- 
ment of  the  latter  (fig.  152). 
If  the  marrow  of  the  bone  be 
preserved,  fine  blood-vessels 
may  be  seen  in  it.  Perhaps 
“perforating  vessels lying  in 
Yolkmann’s  canals  may  be 
noticed,  especially  in  transverse 
sections. 

G.  Cancellated  Bone  (L  and 

H). — In  the  vertical  section  of 
7//  head  of  a long  bone  showing 

fr  J i I iil  -M  i articular  cartilage  a view  of  the 

'll  illtlii  open  lattice-work  will  be  ob- 

tained (Lesson  XIY.  3).  Or  a 
T.S.  may  be  made  across  the 
head  of  a long  bone,  e.g.^  a 
femur,  preferably  of  a young 
animal.  Stain  a section  in 
haematoxylin,  picro-carmine,  or 
eosin-logwood,  and  mount  it  in 
glycerine-jelly. 

(a.)  (L)  Xote  the  network  of 
osseous  trabeculae  (fig.  153)  bounding  the  spaces  or  cancelli.  In 
the  latter  lies  red  marrow,  and  on  their  walls  are  osteoblasts.  In 

the  centre  of  some  of  the 
trabeculae  may  be  seen  a 
deeper  stained  irregular  bar, 
the  remains  of  calcified  carti- 
lage  (fig.  153,  CC).  On  the 
calcified  cartilage  is  deposited 
osseous  tissue. 

(h.)  (H)  In  each  trabecula 
note  the  fibrous  matrix  and 
the  bone-corpuscles.  In  the 
interior  of  some  of  the  tra- 
beculae the  remains  of  un- 
absorbed calcified  cartilage. 
Xote  also  how  the  osseous 
tissue  with  spherical  bulgings  advances  upon  the  calcified  cartilage 
By  carefully  shading  the  light,  it  will  be  seen  that  a more  or  less 


Fig.  152.— L.S.  Injected  Bone.  P.  Perios- 
teum ; B.  Bone ; V.  Blood-vessels. 


T 

Fig.  153.— Cancellated  Bone.  C.  Cancellus ; 
T.  Trabecula;  CC.  Calcified  cartilage;  B. 
Bone ; O.  Osteoblast. 


XIII.] 


BONE,  OSSEOUS  TISSUE,  ETC. 


i8i 


spherical  mass  of  osseous  tissue,  surrounds,  and  in  fact  is  formed  r.y, 
each  bone-corpuscle  (fig.  154).  There  are  thus  spherical  masses — 
cell- territories,  as  it  were 
of 


— and  in  the  centre 
each  a bone-corpusclc. 

7.  Fibrillar  Structure 
of  Lamellse  (H). — De- 
calcify a bone  in  v.  Ebner’s 
fluid  (10-15  cent,  sodic 
chloride  and  1-3  per  cent, 
hydrochloric  acid).  Either 
this  fluid  or  that  given  at 
p.  37  may  be  used.  Scrape 
off  a thin  ] amelia  and 
examine  it  in  water. 

(H)  Observe  the  fibres 
posed  of  fibrils  arranged 


Fig.  154. — SmaTPart  of  FI^.  153,  X300.  CC.  Calcified 
cartilage ; B.  Bone ; O.  Osteoblast ; BO.  Bone- 
corpuscles. 


of  which  it  consists.  They  are  com- 
in  bundles.  They  are  best  seen  near 
the  edge.  Fibres  in  different  planes  cross  each  other  at  a right 
or  obtuse  angle. 


ADDITIONAL  EXERCISES. 

8.  Blood-Vessels  of  Bone. — These  may  be  injected  from  the  descending 
aorta  of  a rabbit  with  a saturated  and  liltered  watery  solution  of  Briicke’s 
Berlin  blue.  One  must  remember  that  the  injection  often  fails.  Inject  from 
the  abdominal  aorta,  after  a time  using  pretty  Ingh  pressure.  It  is  well 
to  ligature  the  inferior  vena  cava  after  some  injection  has  flowed  from  it. 
Decalcify  the  bone  in  chromic  acid.  The  sections  may  be  stained  with  very 
dilute  fnchsin. 

9.  Decalcified  Bone. — Make  a T.S.  of  dense  bone — rather  a thick  section  — 
steep  it  in  alcohol,  transfer  it  to  a slide,  and  mount  it  in  glycerine-jelly,  or  in 
a morsel  of  dry  Canada  balsam.  In  the  latter  case  the  slide  must  be  heated 
to  melt  the  balsam.  Examine  it  at  once,  when  the  lacunae  and  canaliculi  will 
be  seen  black  on  a clear  ground.  They  still  contain  air,  hence  their  black 
appearance.  Gradually,  howevt  r,  the  jelly  penetrates  into  the  canalicular 
system,  and  this  characteristic  appearance  vanishes,  but  it  is  an  instructive 
exercise  for  students  to  perform  this  experiment.  Afterwards  the  lamellae 
remain  quite  distinct  in  the  preparation,  there  are  also  faint  indications  of  the 
canaliculi. 

10.  Bone  in  Polarised  Light. — Examine  a non -decalcified  transverse  section 
of  the  shaft  of  a long  bone  in  polarised  light  (Lesson  XVI.  16).  AVhen  the 
Nicols  are  crossed,  each  Haversian  system  has  a bright  cross  on  a dark  ground, 
while  the  lamellae  are  alternately  bright  and  dark.  The  crosses  are  also  seen 
in  decalcified  bone,  so  that  they  are  produced  by  the  organic  ba^is  or  ossein 
of  the  bone. 


1 82 


PRACTICAL  HISTOLOGY. 


[XIV. 


LESSON  XIV. 

BONE  AND  ITS  DEVELOPMENT. 

Bone  is  developed  either  in  connection  with  cartilage  or  membrane. 
The  former  is  called  endochondral  and  the  latter  intramem- 
branous  ossification.  With  the  exception  of  a part  of  the  skull  — 
its  sides  and  vault — and  nearly  all  the  facial  hones,  all  the  hones 
are  laid  down  in  hyaline  cartilage. 

1.  Development  of  Bone— T.S.  of  Foetal  Bone. — Decalcify  the 
shaft  of  the  femur  or  other  long  bone,  e.g.,  the  radius  and  ulna, 


Fig.  155. — T.S.  Radius  and  Ulna  of  an  Embryo  Dog.  1.  Interosseous  ligame;it ; p.  Peri- 
osteum ; me.  Medullary  cavity ; msp.  Subperiosteal  tissue ; lo.  Osseous  trabecube  ; 
n.  Point  where  the  ligament  enters  the  bone  ; n'.  Union  of  ligament  with  the  perios- 
teum. Observe  that  at  first  the  interosseous  membrane  is  inserted  into  a depression 
in  the  bone  ; when  the  membrane  becomes  ossified  a ridge  is  formed. 


of  a newly-born  kitten  in  picric  acid  (p.  37).  Make  transverse 
sections,  and  stain  them  with  picro-carmine.  Mount  one  in 
Tarrant’s  solution. 

(«•)  (L)  Observe  the  periosteum  (fig.  156,  a,  h),  composed  ex- 
ternally of  connective  tissue,  with  fusiform  corpuscles  stained  red. 
Under  this,  one  or  more  layers  of  cubical  or  somewhat  flattened 
nucleated  cells,  osteoblasts  (c).  They  pass  into  and  line  the  Haver- 
sian spaces,  thus  reaching  the  cancelli  and  medullary  cavity,  which 


XIV.]  BONE  AND  ITS  DEVELOPMENT.  1 83 

they  also  line,  so  that  every  spicule  or  surface  of  young  bone  is 
covered  by  them.  From  them  the  bone-corpuscles  are  formed. 

(b.)  The  osseous  tissue  is  stained  red,  and  forms  an  anastomosing 
series  of  traheculce  bounding  large  spaces — Haversian  spaces — 
containing  blood-vessels  and  marrow,  and  lined  by  osteoblasts. 
Processes  from  the  trabeculae  project  into  the  deeper  layer  of  the 
periosteum.  At  this  stage  a concentric  arrangement  of  the  lamellae 
leading  to  the  formation  of  Haversian  systems  has  not  yet  taken 
place.  In  the  bone  matrix,  the  bone-corpuscles  (/),  irregular,  re- 
fractive, nucleated  cells,  each  lying  in  a lacuna. 

(c.)  (H)  Under  the  periosteum  and  in  the  spaces  may  be  seen 
larger  multinucleated  cells. 

Osteoclasts  or  Myeloplaxes  (fig.  156,  Z ). — The  cells  are  much 
larger  than  the  osteoblasts,  con- 
tain many  nuclei,  and  lie  in  little 
depressions  of  the  bone  eroded 
by  themselves.  These  depres- 
sions are  called  Howship’s 
lacunae.  These  cells  are  con 
cerned  in  the  removal  of  bone. 

2.  Intra-Car tilaginous  For- 
mation of  Bone  (L  and  H). — 

Decalcify  in  picric  acid  the  pha- 
langeal bones  of  a four-months 
foetus.  Make  longitudinal  ver- 
tical sections,  stain  in  picro- 
carmine,  and  mount  in  Farrant’s 
solution. 

(a.)  (L)  At  the  head  of  the 
bone  observe  a mass  of  hyaline 
cartilage  (fig.  157),  and  lower 
down  bone,  and  where  the  two  are  continuous  an  irregular  festooned 
margin,  the  line  of  ossification. 

{h.)  The  cartilage  at  the  upper  })art  is  hyaline,  with  the  cells 
small,  and  arranged  singly  or  in  groups,  while  deeper  down  the 
cartilage-capsules  are  beginning  to  be  arranged  in  rows,  and  below 
this  are  larger  cartilage-capsules  with  clearer  contents,  and  a some- 
what refractive  matrix  between  them. 

(c.)  Under  this,  the  line  of  ossification,  with  its  festooned 
margin,  are  spicules  of  calcified  cartilage  passing  downwards 
towards  the  medullary  cavity.  In  the  bony  part,  the  primary 
medullary  spaces,  with  their  osteoblasts,  blood-vessels,  osteoclasts, 
and  the  newly-formed  bone  deposited  on  the  calcified  cartilage. 

(d,)  The  first  bone  is  formed  under  the  periosteum  (fig.  157),  by 
means  of  the  subperiosteal  osteoblasts.  As  these  osteoblasts 


teum ; c.  Layers  of  osteoblasts,  with 
k.  Osteoclasts ; m.  Matrix  of  bone ; L 
Lacunae,  with  bone-corpuscles. 


PRACTICAL  HISTOLOGY. 


[XIV. 


184 


become  embedded  in  the  bone  matrix  they  become  bone-corpuscles. 
This  piece  of  bone  is  perforated  by  blood-vessels,  which  pass  into 
the  primary  medullary  spaces  in  the  hollo  wed-out  cartilage. 

{e,)  (H)  Examine  the  several  parts.  Search  for  an  osteoclast 
lying  in  a little  cavity — Howship’s  lacuna — and  notice  that  while 
the  margins  of  the  trabeculae,  covered  by  recently-formed  bone, 
are  stained  of  a deep  red  colour  by  the  carmine,  no  such  red  stain 


Osteogenic  tissue 
Endochondral  bone 

Blood-vessel 
Perichondral  bone 

Enlarged  cartilage-capsules 


Enlarged  cartilage-capsules. 
Calcified  cartilage  in  the  form  of 

Pointed  processes  projecting  into 
the 

Primary  medullary  spaces. 
Perichondral  bone. 


Pig.  157. — Dorso-palmar  Longitudinal  Section  of  the  Second  Phalanx  of  the  Finger  of 
a Four-Months  Foetus. 


is  seen  where  the  osteoclast  is  embedded.  It,  in  fact,  is  eroding 
or  eating  away  bone. 

3.  Epiphysis  and  Epiphysial  Cartilage  (H  and  L). — Make 
longitudinal  vertical  sections  of  a young  rabbit’s  femur  or  tibia 
to  show  the  epiphysial  cartilage.  Stain  it  in  picro-carmine  and 
mount  in  glycerine-jelly  ; or,  better  still,  double  stain  it  in  hsema- 
toxylin  and  picro-carmine  or  hsematoxylin  and  eosin.  In  the  last 
case  the  cartilage  will  be  blue,  the  rest  red  or  copper-red.  The 
method  of  double  staining  is  particularly  valuable  for  the  study 
of  bone  development. 

{a.)  (L)  Observe  the  thin  layer  of  encrusting  cartilage  on  the 
head  of  the  bone  (fig.  158,  (7),  and  under  it  the  cancellated  bone 
of  the  head  of  the  tibia.  This  cartilage  is  continuous  below 
with — 

(6.)  A broad  layer  of  cartilage  between  what  is  to  be  the  head 


BONE  AND  ITS  DEVELOPMENT. 


XIV.] 


I8S 


of  the  bone — the  epiphysis  {E) — and  the  future  shaft  or  diaphysis 
of  the  bone  (D). 

(c.)  In  the  epiphysial  cartilage  (^EC)  the  vertical  rows  of  carti- 
lage-cells, smaller  above,  and  larger  and  more  quadrilateral  below 
(fig.  158  and  fig.  159,  (7). 

(d.)  The  shaft  with  longitudinal  spaces — the  primary  medul- 
lary spaces  (fig.  158,  ME) — bounded  by  trabeculae  of  calcified 
cartilage  partly  covered  by  bone  (fig.  159,  h).  The  spaces  con- 
tain young  marrow  (c). 

(e.)  (H)  Study  specially  the  cells  of  the  epiphysial  cartilage 
(fig.  159,  0),  and  notice  that  the  cells  and  capsules  are  smaller 
above  and  larger  below — zone  of  enlarged  cartilage-capsules.  Some 
of  the  enlarged  cartilage- 
capsules  of  the  lowest  row 
may  be  seen  opening  into 
the  primary  medullary 
spaces.  The  line  of  ter- 
mination of  these  spaces  is 
called  the  line  of  ossifica- 
tion. The  bone  grows  in 
length  by  the  proliferation 
of  the  epiphysial  cartilage- 
cells. 

(/.)  Bounding  the  pri- 
mary medullary  spaces, 
directive  trabeculae  of  cal- 
cified cartilage  (h')  with  a 
deposit  of  bony  matter  on 
them.  ISTote  specially  the 
osteoblasts  (/>)  partially  em- 
bedded in  the  osseous  matter  which  they  themselves  secrete  or 
form.  When  they  become  embedded  in  the  osseous  products 
of  their  own  activity,  they  are  then  called  bone-corpuscles.  Kote 
also  that  the  bone  on  the  cartilage  is  bounded  by  convex  surfaces 
which  fit  into  corresponding  depressions  in  the  cartilage.  On 
the  larger  trabeculae  may  be  found  osteoclasts  lying  in  little 
cavities — Howship’s  lacunae — which  they  have  eroded.  The 
spaces  themselves  are  filled  with  red  marrow  (c)  and  blood- 
vessels (v). 

4.  Intra-Membranous  Formation  of  Bone  (H  and  L). — Take  the 
parietal  bone  of  a foetus  when  the  parietal  bone  is  only  partially 
ossified ; scrape  off  the  periosteum  from  a part  near  the  periphery 
of  the  ossified  part ; stain  in  picro-carmine  and  mount  in  Tar- 
rant’s solution. 

(a.)  Observe  at  one  part  the  fibrous  matrix,  and  shooting  from 


Fig.  158.— V.S.  Head  of  Tibia  of  a Young  Habbit, 
greatly  reduced  to  show  details.  C.  Encrust- 
ing cartilage  ; E.  Epiphysis  ; EC.  Epiphysial 
cartilage  ; D.  Diaphysis  ; p.  Periosteum  ; MS. 
Primary  medullary  spaces. 


PRACTICAL  HISTOLOGY. 


1 86 


[XIV. 


it  calcified  fibres 


c 


ftyiapis 


ni/ 


of  connective  tissue,  these  covered  with  osteo- 
blasts (fig.  i6o). 

I,  Marrow  of  Bone. — It  fills 

the  medullary  cavity  of  long 
hones,  the  cancelli  of  spongy 
bone,  and  it  occurs  in  some  of 
the  larger  Haversian  canals. 
There  are  two  varieties,  yellow 
and  red,  the  difference  in 
colour  being  due  to  the  former 
containing  a large  amount  of 
fat-cells. 

Yellow  marrow  occurs  in 
the  medullary  cavity  or  canal 
and  the  larger  cancelli  of  long 
bones..  Besides  a small  amount 
of  connective  tissue  and  blood- 
vessels, it  consists  principally  of 
fat-cells. 

Bed  marrow  (H)  occurs  in 
the  spongy  tissue  at  the  ends  of 
long  bones,  in  the  short  bones 
of  the  hands  and  feet,  flat 
bones  of  the  skull,  epiphyses  of 
long  bones,  clavicle,  ribs,  and 
in  the  medullary  canal  of  the 
long  bones  of  some  animals,  e.g., 
guinea-pig,  rat,  rabbit.  It  con- 
sists of  delicate  connective 
tissue  with  blood-vessels  and  numerous  cells  of  several  varieties. 

(i.)  Medullary  or  Marrow  Cells  (fig.  i6i,  a,  6,  c), — They  are 
^ in  the  most  numerous,  and  are 

Connective  tissue  bundles.  ’ 

nucleated  cells,  not  unlike 
large  leucocytes.  They  are 
spherical,  with  finely  granular 
protoplasm  and  a spherical 
pale  nucleus.  Sometimes 
they  contain  two  nuclei. 
Examined  quite  fresh  in 
serum,  no  nucleus  is  visible, 
but  it  is  revealed  by  the 
action  of  acetic  acid  or 
dilute  alcohol.  The  proto- 
plasm of  some  of  them  contains  numerous  highly  retractile  granules, 
and  that  of  others  some  brownish  granules.  There  is  also  a smaller 


7^/ 


Fig.  159. — L.S.  Head  of  a Metacarpal  Bone  of 
a Babbit,  aged  three  months.  C.  Epiphy- 
sial-cartilage ; 0.  Bone ; a.  Ilow  of  carti- 
lage-cells; h.  Cartilaginous  trabeculae;  m. 
Primary  medullary  space ; n.  Osseous 
deposit;  h'.  Directive  cartilaginous  trabe- 
culae ; p.  Bone-corpuscle  being  embedded 
in  the  osseous  matrix;  c.  Marrow-cells;  v. 
Injected  blood-vessel,  X 240. 


Osteoblasts.  Calcified. 


Non-calcified. 


Fig.  160. — Surface  Section  of  a Parietal  Bone  of  a 
Human  Embryo  x 240. 


BONE  AND  ITS  DEVELOPMENT. 


XIV.] 


187 


variety  of  corpuscle.  They  exhibit  amoeboid  movements  under 
proper  conditions. 

(2.)  Cells  with  Budding  Nuclei  (fig.  161). — Far  less  numerous, 
but  easily  distinguished,  are  large  finely  granular  cells,  each  with 
a single  large,  often  twisted,  nucleus,  which  in  some  looks  as  if 
it  were  composite,  in  others  it  would  seem  to  consist  of  several 
parts  united  by  some 
substance  like  the  nuc- 
leus itself.  There  is 
very  great  variety  in  the 
shape  of  those  nuclei, 
which  are  visible  in  the 
fresh  condition  of  the  \ 
cell.  These  cells  are  yj 
not  amoeboid  (fig.  161, 

/b  0. 

(3.)  Myeloplaxes.  — 

These  are  larger  than 

the  foregoing,  consisting  fig.  161.— Cells  from  the  Bed  Marrow  of  the  Tibia  of  a 
of  a finely  granular 


’(!S) 


protoplasm  with  numer-  hoi ; /,  g.  After  dilute  alcohol ; h,  i Large  cells 
^ ^ with  i»udd:‘  ■ ■ - 


d,  e.  Marrow-cells  after  dilute  alco- 
jr  dilute  alcohol ; i Large 
^ . /n  r \ with  budding  nuclei ; Myeloplaxes,  x 300. 
ous  nuclei  (tig.  101,  m). 

What  relation  there  is  between  (2)  and  (3),  or  if  there  is  any  relation 
at  all,  is  entirely  unknown. 

(4.)  There  are  to  be  found  cells  smaller  than  but  not  unlike  (i), 
with  a homogeneous  protoplasm  which  has  a reddish  tint  and  a 
spherical  nucleus.  Some  of  them  have  a small  bud  at  the  side 
(fig.  1 6 1,  /,  g).  They  are  regarded  as  cells  from  which  coloured 
blood-corpuscles  are  formed.  They  are  regarded  by  Bizzozero  as 
similar  to  the  nucleated  red  blood-corpuscles  of  the  embryo 

(5.)  Always  a few  fat  cells. 

(6.)  Numerous  red  blood-corpuscles  from  the  blood-vessels  in  the 
red  marrow. 

(7.)  Sometimes  osteoblasts  may  be  detached  along  with  the  other 
constituents  of  the  marrow. 

Lay  open  a rib  or  a long  bone  of  a guinea-pig  or  rabbit ; remove 
a little  of  the  red  marrow  and  diffuse  it  in  blood-serum  or  normal 
saline.  With  a high  power  search  for  examples  of  each  of  the 
foregoing  kinds  of  cells.  The  vertebra  of  a calf  may  be  used,  and 
from  it  the  red  marrow  is  readily  expressed  by  squeezing  it  in  a 
vice. 

The  nuclei  in  some  of  the  cells  are  best  revealed  by  the  action  of 
dilute  alcohol. 

5.  Bed  Marrow. — (i.)  Squeeze  out  some  of  the  red  marrow  from 
a rib  of  a guinea-pig  or  rat ; shake  it  in  a test-tube  containing 


i88 


PRACTICAL  HISTOLOGY. 


[XIV. 


normal  saline  tinged  with  methyl-green,  aldehyd-green,  or  aniline- 
green.  The  marrow  will  fall  as  a precipitate.  Examine  a little  of 
this  in  normal  saline.  Stain  some  in  picro-carmine,  and  mount  in 
dilute  glycerine. 

(H)  Observe  the  various  forms  of  marrow  and  other  cells  met 
with,  the  nuclei  tinged  red.  The  large  myeloplaxes  with  numerous 
nuclei  have  their  protoplasm  green  and  the  nuclei  red.  The  fat-cells 
are  quite  green. 

(ii.)  Place  some  of  the  red  marrow  for  12-24  hours  in  dilute 
alcohol,  mount  and  stain  the  cells  with  picro-carmine.  In  this  way 
the  nuclei  of  all  the  cells  are  stained  red,  their  protoplasm  yellowish, 
while  nucleoli  are  revealed. 

(iii.)  Harden  some  of  the  red  marrow  in  Hayem’s  fluid  for 
twenty-four  hours.  Wash  the  deposit,  stain  (picro-carmine)  and 
mount  a little  in  glycerine.  This  is  a good  hardening  medium  for 
this  purpose ; the  red  blood-corpuscles  can  he  readily  distinguished, 
while  the  nuclei  in  all  the  cells  are  distinct. 

6.  T.S.  Red  Marrow. — Small  pieces  of  the  ribs  of  a young 
rabbit  are  placed  in  the  following  fluid  for  one  day  : — 


Rinse  in  water,  and  transfer  to  picric  acid  to  decalcify  the  bone. 
The  tissue  can  then  be  soaked  in  gum,  then  hardened  in  alcohol 
and  sections  cut.  Sections  may  be  stained  with  eosin  and  logwood. 
The  eosin  stains  any  cells  containing  haemoglobin  of  a reddish-orange 
tint. 


7.  Cover-Glass  Preparation  of  Red  Marrow  (H). — As  described  for  blood, 
get  a thin  layer  of  red  marrow  on  a cover-glass  and  stain  it  for  twenty-four 
hours  in  Biondi’s  fluid  (p.  140).  Dry  cover-glass  preparations  may  also  be 
stained  with  methylene-blue,  eosin-hsematoxylin,  or  methyl-green.  Mount 
the  cover  in  xylol-balsam.  If  desired,  clear  it  up  with  oil  of  cloves  in  which 
a little  eosin  is  dissolved,  and  remove  the  clove-oil  by  xylol.  This  method 
yields  excellent  preparations. 

8.  Squeeze  on  a slide  a little  of  the  red  marrow  from  the  rib  of  a young 
rabbit.  Squeeze  a little  between  two  cover-glasses  to  get  in  a thin  film,  and 
expose  the  latter  for  a minute  or  two  to  the  vapour  of  osmic  acid.  Stain  it 
with  picro-carmine  and  mount  it  in  glycerine. 


Sodic  sulphate 
Mercuric  chloride 
Water  . 


2.5  grams. 

0-25  „ 


500  cc. 


ADDITIONAL  EXERCISES. 


XV.] 


MUSCULAR  TISSUE. 


189 


LESSON  XV. 

MUSCULAR  TISSUE. 

Muscle  histologically  occurs  in  two  varieties — (i.)  Non-striped; 
(2.)  Striped.  Non-striped  muscles  are  involuntary,  while  striped 
muscles,  as  a rule,  are  voluntary ; but  the  heart-muscle  is  an 
exception,  for  though  striped  it  is  involuntary. 

Non- Striped  (smooth,  involuntary).  Occurs  in  the  outer  coats 
of  the  lower  half  of  oesophagus ; muscular  coat  and  muscularis 
mucosae  of  stomach  and  intestines ; villi ; ureter,  bladder,  and 
urethra ; pelvis  and  capsule  of  kidney  ; trachea  (trachealis  muscle) ; 
bronchi ; oviduct ; uterus  ; iris,  ciliary  muscle  ; erector  pili  muscles 
of  skin,  sweat  glands ; coats  of  blood-  and  lymph- vessels ; capsule 
and  trabeculae  (lymphatics,  spleen),  and  ducts  of  some  glands,  sali- 
vary, bile-ducts,  &c. 

It  consists  of  nucleated  elongated  fusiform  contractile  cells,  held 
together  by  a clear  cement.  The  cells  taper  towards  their  extremities, 
and  although  they  appear  homogeneous  in  reality  they  seem  to  consist 
of  longitudinally  arranged  fibrils  held  together  by  a sarcoplasm,  or  at 
least  a transparent  material.  The  nucleus  is  oval  or  rod-shaped. 
Some  state  that  the  cells  have  an  elastic  sheath.  Each  cell  is  from 
4 to  ] o (g  - agVrr  in  breadth  and  40-200  - ^4^ 

inch)  in  length. 

1.  Non-Striped  Muscle  (H). — Place  thin  strips  of  the  muscular 
coat  of  the  intestine  for  forty-eight  hours  in  a 25  per  cent,  solution 
of  nitric  acid,  which  softens  the  connective  tissue  and  renders  the 
tissue  yellow.  Wash  it  thoroughly  in  water.  Tease  a small  part 
in  glycerine  or  Earrant’s  solution.  Or  macerate  a small  piece  of 
intestine  in  per  cent,  bichromate  of  potash  for  48  hours.  Cells 
can  then  he  readily  isolated. 

(a.)  Select  an  isolated  fibre.  It  is  spindle-shaped,  elongated,  or 
fusiform,  tapering  to  both  ends,  and  in  its  centre  there  is  an  oval 
nucleus,  distinguished  by  its  being  rather  more  refractive  than  the 
rest  of  the  cell  (fig.  162).  At  the  poles  of  the  nucleus  there  may 
be  a few  granules. 

It  is  very  difficult  to  stain  these  cells  after  the  action  of  nitric 
acid,  but  this  may  be  done  with  magenta,  provided  the  nitric  acid 
be  entirely  washed  out  of  the  tissue  beforehand.  Each  cell  is  said 
to  have  a sheath,  but  that  cannot  be  seen  in  fibres  prepared  in  this 
way. 


190 


PRACTICAL  HISTOLOGY. 


[xv. 


Fig.  162.  — Isolated  Smooth 
Muscular  Fibres.  Nitric 
acid,  X 300. 


2.  Muscle-Cells  from  the  Frog’s  Bladder  (L  and  H).  — Distend 
the  frog’s  bladder  with  dilute  alcohol  thus.  Transfix  the  skin  on 
each  side  of  the  anus  with  two  pins,  and  tie  round  them  a thread  so 
as  to  occlude  the  anus ; open  the  abdomen, 
make  a slit  into  the  large  intestine,  clear  out 
any  residues  it  may  contain,  and  inject  dilute 
alcohol  through  the  intestine  into  the  blad- 
der. When  the  latter  is  full,  ligature  it  at 
the  neck,  and  suspend  it  for  twenty-four 
hours  in  a large  quantity  of  dilute  alcohol. 
Then  open  the  bladder,  and  with  a camel’s- 
hair  brash  pencil  away  all  the  lining  epi- 
thelium. Stain  a portion  of  the  bladder  in 
logwood  and  mount  it  in  balsam,  or  stain 
in  picro-carmine  and  mount  it  in  Far  rant’s 
solution. 

(a.)  Observe  the  thin  membrane,  traversed 
ill  every  direction  by  thicker  or  thinner  tra- 
beculse  of  smooth  muscle  (fig.  163,  a).  Tlie 
trabeculae  consist  of  numerous  long  fusiform 
nucleated  cells  (c) — the  nuclei,  long,  narrow, 
and  oval.  Some  of  the  cells  are  triradiate 
(h).  These  are  what  S.  Mayer  has  called  atypical  cells.  Oval  nuclei 
with  blunter  ends  are  seen  in  the  fibrous  covering  of  the  bladder  {d). 
They  are  the  nuclei  of  connective-tissue  cells. 

3.  T.S.  Non-Striped  Muscle 
(H). — This  is  obtained  by  mak- 
ing transverse  sections  of  the 
circular  muscular  coat  of  the 
small  intestine  (cat),  previously 
hardened  in  chromic  acid  and 
spirit,  or  Muller’s  fluid.  Stain 
a section  in  hsematoxylin  and 
mount  in  balsam. 

(a.)  Observe  polygonal  areas 
of  unequal  size,  mapped  out 
from  each  other  by  a refractive 
(fig.  164)  cement  substance. 
Each  area  corresponds  to  the 
transverse  section  of  a fibre. 
Some  of  the  areas  contain  a 
nucleus  {n),  others  not.  Sur- 
rounding groups  of  these  areas  are  fine  septa  of  connective  tissue 
(s*),  which  map  out  the  fasciculi  or  bundles  of  cells.  The  fibres  are 
arranged  in  bundles  or  fasciculi,  each  surrounded  by  an  envelope  of 


Fig.  163.— Bladder  of  Frog.  a.  Large  strands 
of  smooth  muscle  ; h.  Triradiate,  and  c. 
Fusiform  muscle-cells ; d.  Nuclei  of  con- 
nective-tissue corpuscles. 


MUSCUI.AE  TISSUE. 


XV.] 


19  A 


A.  T.S.  non-striped  muscle,  in- 
testine of  cat;  B.  Longitudinal  strip  of 
intestine  of  rabbit;  c.  Cell;  n.  Nucleus; 
s.  Septum  of  connective  tissue.  Chro- 
mic and  bichromate  fluid,  haematoxylin. 


connective  tissue.  The  fasciculi  are  large  or  small  according  to  the 
number  of  fibres  entering  into  their  composition. 

(b,)  In  some  preparations  one  can  see  intercellular  bridges  between 
adjacent  cells,  like  those  that  occur  in  squamous  epithelium. 

4.  L.S.  of  Non-Striped  Muscle  (H). — This  may  be  obtained  by 

making  a longitudinal  section  of  . 1 . 

the  lougitudinal  coat  of  the  in- 
testine,  but  it  is  better  to  strip 
off  a thin  lamella  of  this  coat  from 
the  intestine  of  a rabbit  hardened 
in  spirit  or  Muller’s  fluid.  Stain 
in  hsematoxylin  and  mount  in 
balsam. 

(a.)  Observe  the  oval  fusiform 
nuclei  (fig.  164,  n)  lying  in  narrow 
fusiform  areas — the  cells.  The 
boundary-lines  between  the  cells 
are  usually  not  well  defined. 

5.  Cement  Substance  of  Non- 
Striped  Muscle  (H). — With  dis- 
tilled water  wash  out  the  contents 
of  the  small  intestine  of  a freshly-killed  rabbit,  or  the  large  intes- 
tine of  a frog.  Tie  one  end  of  the  gut,  and  fill  it  with  .5  per  cent, 
solution  of  silver  nitrate,  and  tic  the  other  end  of  the  gut.  Suspend 
the  whole  in  \ per  cent,  solution  of  silver  nitrate  for  ten  minutes 
or  so.  Slit  up  the  gut  along  the  line  of 
attachment  of  the  mesentery.  Wash  it 
in  water  and  expose  it  to  light.  It  soon 
becomes  brown.  • Lay  it  on  a glass  plate, 
mucous  surface  uppermost,  and  with  a 
scalpel  scrape  away  all  the  mucous  and 
submucous  coat,  which  is  very  easily 
done,  especially  if  the  intestine  has  been 
macerated  for  about  twenty-four  hours  in 
water.  There  remain  only  the  muscular 
and  thin  serous  coats.  Harden  in  alco- 
hol. Snip  out  a piece,  dehydrate  com- 
pletely in  absolute  alcohol,  and  mount  in  Fig.  165.— Cement  Substance  of 

Smooth  Muscle,  Intestine  of 

Oaisam.  Babbit.  Silver  nitrate. 

(a.)  Observe  the  narrow  elongated 
fusiform  areas  bounded  by  silver  lines  (fig.  165);  they  indicate  the 
outline  of  the  fusiform  cells.  On  focussing  upwards  and  down- 
wards, notice  the  longitudinal  and  circular  direction  of  the  fibres 
crossing  each  other.  In  this  preparation  also  many  lymphatic 
paths  lined  by  sinuous  epithelium  may  be  seen.  They  are  recog- 


192 


PRACTICAL  HISTOLOGY. 


[xv. 


nised  by  the  dilatations  in  their  course,  and  by  the  character  of  the 
endothelial  cells  lining  them.  By  focussing  deeply,  the  silver  lines 
of  the  endothelium  of  the  serous  membrane  may  be  seen. 

6.  Methylene-Blue  Method  (S.  Maf/er). — Into  the  blood-vessels 
of  a cat — e.g.,  from  the  aorta — inject  the  following  solution  : — 

Methylene-blue  (S.  Mayer’s)^  . . . . i gram. 

Normal  saline  .......  300  cc. 

After  an  hour  or  so,  open  the  abdomen ; the  intestines  appear 
blue ; cut  out  a portion  of  the  muscular  coat  and  place  it  in  the 
following  mixture  : — 

Picro-Glycerine  Mixture. 

Saturated  watery  solution  ammonium  picrate  . 100  cc. 

Glycerine  .......  100  ,, 

On  teasing  a small  piece  of  the  muscular  coat  in  the  same  mixture, 
isolated  muscle  cells  are  readily  found,  the  nucleus  being  stained  of 
a faint  rosy-pink. 


ADDITIONAL  EXERCISES. 

7.  Fibrillar  Plexus  in  Muscle-Cells  (H). — Kill  a newt,  open  its  abdomen, 
and  pin  out  its  intestine  and  mesentery  on  a thin  piece  of  cork  with  a hole  in 
it  corresponding  to  the  mt'sentery.  Place  it  for 
twenty-four  hours  in  a 5 per  cent,  solution  of  am- 
monium chromate.  After  this  wash  away  all  the 
chromate,  stain  a piece  of  the  mesentery  in  logwood, 
and  mount  it  in  balsam. 

(a.)  In  the  membrane  observe* narrow  strands  of 
non-striped  muscle  composed  of  very  large  fusiform 
cells.  In  the  large  nuclei  a plexus  of  fibrils,  while  a 
leash  of  fine  fibrils  will  be  seen  in  the  perinuclear  part 
stretching  from  the  poles  of  the  nucleus  to  the  ends  of 
the  fibre. 

8.  Fibrils  in  Smooth  Muscle.  —These  may  be  seen 
for  a short  time  by  macerating  small  pieces  of  the 
stomach  of  a frog  for  twenty-four  hours  in  dilute 
alcohol,  or  in  8-10  per  cent,  of  sodic  chloride  {Engel- 
mann).^ 

9.  Isolated  Smooth  Muscle-Cells. — To  get  these 
cells  quickly  macerate  a small  piece  of  the  muscular 
coat  of  the  stomach,  intestine,  or  bladder  in  33  per 
cent,  caustic  potash  for  15-20  mins.  Tease  and 
observe  in  the  same  solution.  Water  must  not  be 

added,  else  the  effect  of  weak  caustic  alkalies  is  produced,  viz.,  solution  of 
the  cells. 

^ To  be  obtained  from  Dr.  Grubler,  Leipzig,  or  Bindscheidler  k Busch, 
Basel.  ^ PflilgeEs  A rch. , xxv. 


Fig.  166.  — T.S.  Smooth 
Muscle  of  a Cat’s  Intes- 
tine. Chromo  - aceto  - 
osmic  acid.  Shows  in- 
tercellular bridges,  and 
at  a an  artificial  slit, 
X 1000. 


XVI.] 


STRIPED  OR  STRIATED  MUSCLE. 


193 


10.  Grooving  on  Smooth  Muscular  Fibres. — Harden  the  muscular  coat  of 
the  small  intestine  of  a cat  in  chromic  and  bichromate  fluid  or  in  Flemming’s 
fluid.  Stain  and  cut  sections  in  paraffin.  (H)  Observe  the  polygonal  areas, 
but  note  that  fine  intercellular  bridges  connect  adjacent  cells  (fig.  166). 
The  surface  of  each  cell  seems  to  be  grooved  with  canals,  but  what  these  canals 
contain  is  not  known.  In  each  cell  the  cut  ends  of  fine  fibrils  are  seen. 


LESSON  XVI. 

STRIPED  OR  STRIATED  MUSCLE. 

Striped  Muscle — sometimes  called  voluntary  or  skeletal  muscle 
— occurs  in  the  muscles  of  the  skeleton,  pharynx,  upper  half  of 
the  oesophagus,  diaphragm,  the  sphincter  of  the  bladder,  external 
anal  sphincter,  and  the  muscles  of  the  outer  and  middle  ear. 
The  muscular  fibres  of  the  heart  are  also  striped,  but  they  are 
involuntary. 

A Muscular  Fibre  is  cylindrical  in  form,  and  tapers  at  its 
extremities.  They  vary  in  breadth  from  10  to  50  /x  (^4Vo’“T8o‘ 
inch),  but  they  are  broadest  in  the  muscles  of  the  extremities. 
The  fibres  are  i to  i J inches  in  length. 

A muscular  fibre  consists  of  the  following  parts  : — 

(i.)  Sarcolemma,  or  sheath. 

(2.)  Sarcous  substance,  which  is  transversely  striated. 

( 3 . ) Muscle-corpuscles. 

1.  The  Sarcolemma  (H). — (i.)  To  avoid  the  effect  of  the  con- 
tractility of  the  muscle,  kill  a frog  several  hours  before  it  is 
required.  Dissect  out  the  sartorius  muscle,  because  it  is  com- 
posed of  parallel  fibres.  Tear  off  a thin  strip,  and  with  needles 
tease  it  in  didilled  water. 

(a.)  Observe  the  cylindrical  shape  of  the  fibres,  marked  trans- 
versely by  alternate  light  and  dim  stripes.  Run  the  eye  along 
the  edge  of  a fibre,  and  perhaps  a clear,  transparent  bulla  or  bleb 
will  be  seen.  If  so,  it  is  the  sarcolemma  raised  from  the  subjacent 
sarcous  substance  by  water  diffusing  into  the  fibre  (fig.  167,  A). 

(6.)  The  sarcolemma  is  a clear,  transparent,  colourless,  homo- 
geneous elastic  membrane,  forming  a tubular  sheath  for  the  sarcous 
substance.  It  is  allied  to,  but  not  identical  with,  elastic  tissue. 
It  is  much  tougher  and  less  easily  ruptured  than  the  sarcous  sub- 
stance which  it  contains. 

(ii.)  A much  better  plan  is  the  following : — Tease  out  a few 
18  N 


194 


PRACTICAL  HISTOLOGY. 


[XVI. 


fresh  muscular  fibres  on  a slide  in  normal  saline,  and'  across  the 
direction  of  the  fibres  place  a hair.  Cover,  and  press  the  cover-glass 
down  firmly  on  the  fibres.  The  hair  ruptures  the  sarcous  sub- 
stance, which  retracts  and  leaves  the  tougher  unbroken  sarcolemma 
between  the  ends  of  the  ruptured  fibre.  Kemove  the  hair,  and 
the  now  empty  sarcolemma  will  be  seen  (fig.  167,  C). 

(iii. ) Another  excellent  method  is  to  leave  the  muscle  in  a 
saturated  solution  of  ammonium  carbonate.  The  sarcolemma  will 


Fig.  167. — A.  striped  muscle  of  frog,  sarcolemma  raised'  in  the  form  of  a bleb;  B. 

Ruptured  fibre  with  sarcolemma ; C.  Fibre  ruptured  by  a hair ; D.  Effect  of  acetic 

acid  on  a muscle-fibre  ; E.  Muscle-discs.  Ammonium  carbonate. 

be  seen  raised  for  long  distances  from  some  of  the  fibres,  with  a 
little  of  the  sarcoglia  sometimes  adhering  here  and  there  to  its 
under-surface. 

2.  Muscle-Corpuscles,  or  Nuclei  (H). — Tease  a piece  of  a fresh 
frog’s  muscle,  and  irrigate  it  with  2 per  cent,  acetic  acid.  I have 
often  found  the  nuclei  beautifully  stained  in  a piece  of  muscle  that 
has  been  in  ammonium  carbonate  and  then  in  picro-carmine. 

(a.)  Observe  the  sarcous  substance  becoming  swollen  up  and 
more  homogeneous,  while  a number  of  fusiform,  somewhat  shrivelled 
or  shrunken  nuclei,  with  their  long  axis  in  the  long  axis  of  the 
fibre,  come  distinctly  into  view.  ’They  are  now  slightly  more 
refractive  than  the  altered  sarcous  substance,  hence  they  are  seen 
with  greater  distinctness.  Focus  carefully,  and  note  that  these 
nuclei  lie  not  only  under  the  sarcolemma,  but  also  in  the  substance 
of  the  fibres.  Had  a mammalian  muscle  been  used  instead  of  one 
from  an  amphibian — in  the  case  of  most  muscles — the  nuclei  would 
have  been  found  directly  under  the  sarcolemma  only.  The  presence 
of  the  nuclei  is  merely  revealed  by  the  action  of  the  acid,  which 
alters  the  refractive  index  of  the  sarcous  substance,  and  thus  brings 
the  nuclei  into  view  (fig.  167,  D).  Sometimes  faint  longitudinal 
striation  is  exhibited  by  such  a fibre. 

3.  Isolated  Muscular  Fibres. — (i.)  Pith  a frog,  and  plunge  it  in 
a beaker  of  water  at  55°  C.  Leave  it  in  the  water,  and  allow  the 


XVI.] 


STRIPED  OR  STRIATED  MUSCLE. 


^95 


water  to  cool  gradually  {Ranoier).  It  will  now  be  found  that  the 
fibres  of  any  muscle  can  be  dissociated  with  great  ease  ; the  muscles 
to  be  preserved  in  70  per  cent,  alcohol  until  they  are  required. 
By  careful  manipulation  very  long  fibres  may  be  isolated  from  the 
sartorius.  These  muscular  fibres  exhibit  the  ordinary  characters 
of  striped  muscle.  This  is  by  far  the  easiest  method  of  obtaining 
isolated  fibres. 

(ii.)  Place  small  pieces  of  a fresh  muscle  in  the  following  mixture. 
Nitric  acid  saturated  with  potassic  chlorate.  There  must  be  crystals 
of  the  latter  in  the  fluid.  The  tube  or 
vessel  is  speedily  filled  with  yellow 
nitrous  fumes.  It  is  usually  advised 
to  leave  the  muscle  several  hours  in 
this  fluid.  I find,  however,  if  this  be 
done,  that  the  muscle  is  dissolved. 

Half  an  hour  is  usually  sufficient. 

With  glass  rods  remove  the  now 
softened  orange-coloured  muscle,  and 
place  it  in  water.  It  becomes  whitish. 

Shake  it  in  a tube  with  water.  The 
fibres  fall  asunder  quite  readily,  and 
after  having  all  the  acid  removed  by 
prolonged  washing,  they  can  be  stained 
and  mounted. 

4.  Fibrillse  of  a Muscular  Fibre 
(H).  —Place  a frog’s  or  mammal’s 
muscle  in  water  (two  hours),  and  after- 
wards in  dilute  alcohol  for  twenty-four 
hours.  Tease  a small  fragment  of  the 
now  softened  muscle  in  glycerine,  or 
tease  a fresh  muscle  of  a calf  in  white 
of  egg.  If  a fibre  be  split  up,  bundles 
of  fibrils  may  be  seen  as  in  fig.  168. 

Select  a fibre,  and  note  that  at  its 
free  end  it  splits  up  longitudinally  into 
a large  number  of  very  fine  fibrils  or  fibrillae,  each  of  which  is 
transversely  striated  like  the  original  muscular  fibre.  Much  larger 
fibrils  are  obtained  from  the  muscles  of  insects,  e.g.^  Hydrophilus  or 
Dytiscus  (fig.  1 71). 

5.  Muscle-Discs  (H). — The  usual  directions  for  obtaining  these 
are  to  place  dead  muscle  for  several  days  in  dilute  (.2  per  cent.) 
hydrochloric  acid.  The  muscular  fibre  then  cleaves  transversely. 
I have  not  found  this  to  be  a very  satisfactory  method.  A much 
better  plan  is  to  place  small  pieces  of  the  muscle  in  a saturated 
solution  of  ammonium  carbonate  for  several  hours. 


Fig.  t68.— Part  of  a Striped  Muscu- 
lar Fibre  of  a Calf,  teased  in 
white  of  egg,  and  showing  iso- 
lated bundles  of  fibrils,  x 200. 


196 


PRACTICAL  HISTOLOGY. 


[xvi. 


(a.)  Not  only  will  the  s^Ircolemma  be  seen,  but  inside  it,  here 
and  there,  the  sarcous  substance  will  be  seen  cleft 
transversely  into  discs  (fig.  167,  E). 

6.  Ending  of  Muscle  in  Tendon. — This  is  readily 
seen  by  taking  the  lower  end  of  the  sartorius  with 
its  tendon  from  a frog  treated  as  in  Lesson  XVI.  3, 
(i.),  and  which  has  been  afterwards  placed  in  70  per 
cent,  alcohol.  Tease  out  the  muscular  fibres,  when 
their  conical  ends  will  be  seen  ending  abruptly,  and 
the  small  tendons  beginning  as  abruptly  (fig.  169). 

7.  Crab’s  Muscle  (Sarcous  Substance)  (H). — 
Plunge  the  living  muscles  of  a crab — or  better  still, 
use  a stag-beetle — into  absolute  alcohol  for  twenty- 
four  hours.  Stain  a fragment  in  dilute  eosindicema- 
toxylin^  and  mount  it  in  Far  rant’s  solution  or  balsam. 
In  the  latter  case  the  clarifying  reagent  must  contain 
a little  eosin  to  restore  the  eosin  colour  to  the  pre- 
paration. 

(a.)  Observe  the  fibre  striped  as  in  ^ 

fig.  170,  with  alternate  light  and 
dim  discs,  the  dim  discs  (a)  stained 
of  a logwood  tint,  while  the  light 
discs  are  of  a faint  eosin  tint. 

{h.)  In  the  light  disc  {h)  observe  a fine  line  or 
series  of  dots  running  transversely,  sometimes  called 
Dobie’ s line  or  intermediate 
line  (figs.  170,  b,  171,  c), 
dividing  the  light  disc  into 
two  equal  parts,  these  ad- 
hering to  the  ends  of  the 
dim  disc.  They  are  then 
called  lateral  discs. 

(c.)  The  dim  disc  may 
exhibit  slight  vertical  stria- 
tion,  indicating  a tendency 
to  cleave  longitudinally.  If 
transverse  cleavage  be  as- 
sociated with  simultaneous 


Fig.  169.  — Rela- 
tion of  a Tendon 
T.  to  its  Muscu- 
lar Fibre, the  lat- 
ter with  a Coni- 
cal Termination. 


»an(TOniia^im 

BtrtftI:  ramifttfi  niirrtf  inmEU  ce, 

aSprwTMnniin  fi  mirifl  liiiffffB 
ffiraiiLraiiiiaiinrraliiii  1 1 n ifiiiuTafflUJnniiiiiBiin  uj 

Jj^Rfjrn-iimrrrifiW’TiTtLiim 



Mlfemtapm 

iii  ciil  iumiTni 

»ni n - - 

ilsiissssgi^ 


Fig.  170.— Muscular  Fibre  of  longitudinal  cleavage,  small 

Great  Adductoi  of  Rabbit,  n plpTnPTvtc:  orp  oblniriPfl 

Living  and  Extended,  a.  elements  are  ODtaineQ, 

Dim  disc;  h.  Light  disc;  which  \vere  Called  “sarcous 
c.  Intermediate  or  Dobie’s  ??  i t:> 

line;  n.  Nucleus  seen  in  elements  by  Bowiuan. 
profile.  Examined  in  its 

own  juice,  X 300.  a • p 

A Similar  preparation  oi 


Fig  1 7 1.  — Fibril 
of  Muscle  of 
Hydrophilus.  a. 
Dim,  c.  Light 
disc ; h.  Inter- 
mediate line,  X 
2000.  Picrocar- 
mine  and  formic 
glycerine. 


(d.)  Nuclei  may  be  seen, 
similar  preparation  of 
an  insect’s  muscle  may  be  stained  in  picro-carmine.  Tease  it  so 


STRIPED  OR  STRIATED  MUSCLE. 


197 


XVI.] 


as,  if  possible,  to  isolate  a fibril.  Mount  in  formic  glycerine.  The 


A B 


Fig.  172. — Crab’s  Muscles, 
partly  schematic.  A. 
Non-stretched  ; B.  Ex- 
tended; F.  Fibrils;  B. 
Dim  ; L.  Light  stripes. 


Fig.  173.— T.S.  Muscle,  with  its  Sheaths.  P. 
Perimysium ; E.  Endomysium. 


dim  disc  is  stained  red  (fig.  170,  a),  and  if  the  fibre  be  stretched 


Fig.  174.— d.  T.S.  muscle  fibres  of  newt ; B. 
Of  mammal ; C.  T.S.  muscular  fibres 
(mammal),  with  endomysium;  b.  Blood- 
vessel; F.  Fasciculus;  P.  Perimysium. 


Fig.  175. — Capillary  Plexus  in  Muscles. 
a.  Artery;  v Vein;  c.  Capillaries, 
X 250. 


the  details  of  its  structure  can  be  better  seen,  especially  if  a very 
high  objective  be  used  (fig.  171). 


198 


PRACTICAL  HISTOLOGY. 


[XVI. 


8.  T.S.  of  Muscle. — Make  transverse  sections  of  a small  mamma- 
lian muscle  which  has  been  kept  stretched  and  hardened  in  0.5  per 
cent,  chromic  acid  and  afterwards  in  alcohol.  Stain  one  in  logwood 
and  mount  it  in  Canada  balsam. 

(a.)  (L)  Observe  the  sheath  of  connective  tissue  or  perimysium 
surrounding  the  whole  muscle,  and  that  from  it  septa  pass  between 
groups  or  fasciculi  of  the  muscle-fibres,  and  also  a small  amount 
between  the  muscle-fibres,  forming  the  endomysium  (figs.  173, 

174)- 

The  ends  of  the  muscular  fibres  somewhat  polygonal  or  rounded, 
with  stained  nuclei  (one,  two,  or  three)  immediately  under  the 
sarcolemma.  In  amphibian  muscles  (fig.  174,  A)  and  in  a few 

mammalian  muscles,  e.g.^ 
the  semi-tendinosus  of 
the  rabbit,  nuclei  also 
occur  within  the  sarcous 
substance. 

(6.)  (H)  The  ends  of 
the  fibres  appear  finely 
dotted  with  clear  inter- 
spaces, the  dots  corre- 
sponding with  the  ends 
of  the  bundles  of  fibrils 
or  muscle  - prisms  or 
sarcostyles,  while  the 
clear  areas  are  due  to 
what  is  called  sarcoglia. 

9.  L.  S.  Injected 
Muscle  (L).  — Make  a 
longitudinal  section  of 
an  injected  muscle,  i.e,^ 
parallel  to  the  direction 
of  its  fibres.  It  is  better 
to  inject  the  whole  of 
the  posterior  half  of  the 
body,  e.y.,  of  a rabbit, 
from  the  aorta.  The 
will  yield  injected  muscle. 


Fig.  176. — T.S.  and  L.S.  Injected  Striped  Muscle, 
and  2.  T.S.  Muscular  fibres  ; 3.  L.S.;  a.  Arteries. 

preparation  made  for  injected  bone 


Mount  it  in  balsam. 

(a.)  Oliserve  the  elongated  quadrilateral  meshes  of  capillaries 
between  the  muscular  fibres,  but  outside  the  sarcolemma,  and  that 
capillaries  run  between  the  fibres  with  short  transverse  connecting 
branches.  Trace  their  origin  from  an  artery  and  their  termination 
in  a vein  (fig.  1 75). 

10.  T.S.  Injected  Muscle. — Moq^nt,  either  strained  or  unstrained, 


XVI.] 


STRIPED  OF  STRIATED  MUSCLE. 


199 


in  balsam.  In  this  the  cut  ends  of  the  capillaries  between  the 
fibres  will  be  seen  (figs.  1765  177). 

It  is  to  be  noted  that  there  is  a difference  in  the  T.S.  of  con- 
tracted and  uncontracted  muscles  {Spatleholz),  Beautiful  figures 
are  given  in.^ 

11.  L.S.  Eed  Muscle  of  Rabbit  Injected  (L). — Use  the  semi- 
tendinosus  or  soleus  of  a rabbit.  This  shows  the  same  general 
arrangement  of  the  blood-vessels,  but  some  of  the  transverse 
branches  and  some  of  the  veins  have  small  dilatations  or  ampullae 
upon  them,  while  the  capillaries  are  usually  more  tortuous  than 
those  of  the  pale  muscles. 


CARDIAC  MUSCLE. 

12. — Harden  small  pieces  of  the  heart  in  20  per  cent,  nitric 
acid , (forty-eight  hours),  or  2 per  cent,  potassic  bichromate  or 
ammonium  chromate  for  thirty-six  to  forty-eight  hours.  Tease  a 
small  piece  in  Tarrant’s  solution.  Small  pieces  of  muscle  cardiac 
placed  fresh  in  picro-carmine  for  several  days  show  the  structure 
well  when  mounted  in  glycerine. 


Fig.  177. — T.S.  Muscle  In- 
jected. M.  Muscle, 
with  n.  Nuclei,  6.  Blood- 
vessel (capillaries). 


Fig.  178.— Muscular  Fi- 
bres of  the  Human 
Heart. 


Fig.  179.— T.S.  of  a 
Fresh  Frozen  Muscu- 
lar Fibre,  showing 
Cohnheim’s  areas. 


{a.)  Observe  the  faintly  transversely-striated  fibres  made  up  of 
short  quadrilateral  pieces  with  short  oblique  processes,  which  join 
other  musTjular  fibres.  The  muscle-cells  branch  and  anastomose. 
A rather  indistinct  line  of  clear  cement  joins  the  ends  of  adjacent 
cells. 

(h.)  There  is  no  sarcolemma,  but  a well-defined  nucleus  lies  in 
the  substance  of  the  fibre,  while  the  transverse  striation  is  much  less 

^ “ Die  Vertlieilung  d.  Bliitgefasse  im  Musk  el,”  Ahhand.  d.  math.  phys. 
classed,  k.  Sdchsig.  Gescll.  d.  Wissensch.,  1888. 


200 


PRACTICAL  HISTOLOGY. 


[XVI. 


distinct  than  in  skeletal  muscles  (fig.  178).  In  a transverse  section 
of  a cardiac  muscle  the  nucleus  lies  in  the  centre  of  the  fibre,  and 
radiating  from  it  are  fine  lines  (fig.  207) ; an  appearance  somewhat 
similar  to  this  is  shown  in  T.S.  of  insects  muscles. 


ADDITIONAL  EXERCISES. 

13.  T.S,  Frozen  Muscle  (H). — With  a freezing  microtome  make  a transverse 
section  of  a muscle  taken  from  a recently-killed  animal. 

{a.)  The  ends  of  the  fibres  are  mapped  out  into  a large  number  of  small 
polygonal  areas — Cohnheim’s  areas — separated  from  each  other  by  a clear  net- 
work of  lines.  The  darker  areas  correspond  to  the  ends  of  a bundle  of  fibrils — 
the  so-called  muscle-prisms,  while  the  clear  material  between  them  is  the 
sarcoglia  (fig.  179). 

14.  Living  Muscle  (H). — Remove,  with  as  little  injury  as  possible,  some  of 
the  muscular  fibre  from  the  leg  of  a water-beetle  (Dytiscus  or  Hydrophilas), 
place  the  muscle  on  a slide  without  the  addition  of  any  other  fluid,  and  cover  it. 
Examine  it  as  quickly  as  possible,  and  with  the  highest  available  objective. 
In  insects’  muscles  there  is  far  more  protoplasmic  matter  or  sarcoplasm  between 
the  muscle  prisms  or  sarcostyles  than  there  is  in  vertebrate  muscle. 

{a.)  Observe  the  alternate  cross  stripes,  some  of  which  will  be  distinctly  seen, 
while  at  other  parts  they  may  be  very  close  together,  or  the  fibre  may  exhibit 
contraction  waves. 

The  dim  disc  or  band  may  exhibit  slight  longitudinal  striation,  while  a 
dotted  line — Dohies  line — will  be  seen  running  across  the  bright  or  light  disc, 
dividing  it  into  two  so-called  lateral  discs. 

The  nuclei,  surrounded  by  a small  quantity  of  ])rotoplasm,  may  be  visible. 

15.  Crab’s  Muscle  (Methyl- Violet). — Stain  a fragment  of  a crab’s  muscular 
fibre  (hardened  in  alcohol  or  Muller’s  fluid  and  spirit,  p.  29)  with  methyl- violet 
as  described  for  fibrin  (Lesson  III.  18).  Decolorise  it  with  Lugol’s  solution  of 
iodine  in  iodide  of  potassium,  clarify  it  in  aniline-oil  and  xylol,  and  mount  it 
ill  balsam.  Use  all  the  precautions  detailed  under  Weigert’s  method  for  fibrin. 
In  successful  portions,  the  dim  disc,  and  it  alone,  will  be  obtained  of  a deep 
violet. 

Do  this  with  a contracted  fibre,  one  extended,  and  one  relaxed.  In  the 
extended  fibre  observe  that  it  is  chiefly  the  light  disc  which  has  been  elongated 
by  the  extension  (fig.  172,  B).  In  the  contracted  muscle,  the  discs  are  closer 
together  and  narrower,  while  the  fibre  is  broader  at  the  contracted  part. 

16.  Polariscope. — An  ordinary  microscope  can  be  fitted  with  a polariscope, 
which  consists  of  two  Nicols  jirisms  ; one  is  placed  below  the  object,  and  is 
called  the  polariser  (fig.  180),  while  the  other,  the  analyser,  is  placed  above 
the  ocular. 

The  light  reflected  from  the  mirror  as  it  passes  through  the  polariser  is 
polarised.  With  the  analyser  in  position,  look  into  the  ocular,  and  slowly 
turn  the  analyser.  The  best  forms  are  provided  with  a graduated  circle  to 
indicate  the  extent  of  the  rotation.  There  are  two  positions  of  the  analyser  in 
which  the  field  is  quite  dark,  caused  by  the  polarised  rays  being  cut  off.  This 
occurs  when  the  ]danes  of  polarisation  of  the  two  prisms  are  at  right  angles  to 
each  other,  i.e.,  when  the  Nicols  are  crossed.  Between  these  two  positions  of 
the  analyser  a greater  or  less  amount  of  polarised  light  is  transmitted.  Certain 
transparent  histological  preparations  when  placed  on  the  stage  of  the  micro- 


STRIPED  OR  STRIATED  MUSCLE. 


201 


xvl] 


scope  are  dark  when  the  Nicols  are  crossed,  others  under  the  same  conditions 
cause  the  light  to  reappear,  and  appear  bright  on  a dark  field.  They  are  said 
to  be  doubly  refractive.  The  dim  disc  is  doubly  refractive  or  anisotropous^ 
while  the  light  disc  is  singly  refracted,  and  is  isotropous. 

Either  a preparation  of  fresh  muscle  or  a balsam  preparation  may  be  used. 
It  is  well  to  take  a muscle  v/bich  has  broad  and  distinct  stripes  to  see  the 
phenomena,  one  set  of  bands  bright  and  refractive,  and  the  others*  dark  on  a 
dark  ground,  ^.6.,  with  crossed  Nicols. 

Instead  of  a glass-cover  slip,  cover  the  preparation  with  a thin  slip  of  mica, 
or  place  a thin  plate  of  gypsum  under  a preparation  of  striped  muscle.  The 
field  shows  various  colours,  red,  pink,  green,  &c.,  according  to  the  thickness  of 
the  mica  plate,  the  position  of  the  Nicols,  and  the  relation  of  the  axis  of  the 
mica  to  that  of  the  Nicols.  Suppose  the  general  tint  of  the  field  to  be  pink, 
then  any  doubly  refractive  substance  assumes  a tint  complementary  to  the  pink, 
i,e.j  of  a greenish  hue.  On  turning  the  analyser,  the  tint  of  the  field  varies, 


A B 

Fig.  i8o.— a.  Polariser  to  fit  into  Zeiss’s  large  stand  in  a frame  under  Abbe’s  condenser  ; 
B.  Section  of  A showing  arrangement  of  the  prisms. 


and  with  it  the  colour  of  the  anisotropous  substance,  while  the  isotropous  sub- 
stance, being  singly  refractive,  and  having  no  effect  on  the  direction  of  the 
polarised  ray,  has  the  same  colour  as  the  field. 

The  doubly  refractive  property  is  possessed  by  bone  (p.  i8i),  smooth  muscle, 
and  the  white  fibres  of  connective  tissue. 

17.  Obliquely  striated  Muscle  of  Anodon. — Place  a wedge  between  the 
partially  opened  valves  of  Anodon,  the  fresh-water  mussel,  so  as  to  put  the 
fibres  of  the  posterior  adductor  on  the  stretch.  The  whole  animal  may  then 
be  placed  in  dilute  alcohol,  or  i per  cent,  potassium  bichromate,  for  two  days. 
On  teasing  a portion,  isolated  fibres  showing  oblique  striation  are  obtained. 
Or  the  muscle  may  be  examined  fresh  in  sea-water  or  in  the  blood  of  the 
animal. 

Retro-lingual  Membrane  of  Frog  {Ranvicr).^ — Under  the  tongue  of  the  frog 
is  a lymph-sac  which  is  separated  from  the  buccal  cavity  by  a thin  membrane. 
It  contains  striped  muscular  fibres  which  anastomose  with  each  other,  thus 
forming  a plexus — in  this  respect  presenting  a peculiarity.  Cut  ofl*  the  head, 
pull  out  the  tongue,  and  remove  the  membrane.  It  can  be  floated  in  salt 
solution  to  see  it.  Place  the  membrane  for  twenty-four  to  forty-eight  hours 
in  dilute  alcohol,  then  pencil  away  the  epithelium,  and  place  it  for  twenty- 

^ Comptes  RenduSf  1890. 


19 


202 


PRACTICAL  HISTOLOGY. 


[XVII. 


four  hours  in  dilute  methyl-violet-5  B.  Wash  and  mount  in  glycerine.  The 
elastic  fibres  are  stained  of  a bright  blue,  and  they  seem  to  spring  from  the 
ends  and  sides  of  the  muscular  plexus — also  stained  blue.  The  elastic  fibres 
are  fixed  to  the  sarcolemma — which,  however,  is  not  stained  by  the  methyl- 
violet — and  the  union  is  a very  firm  one.  Thus  the  muscular  fibres  divide, 
and  arc  in  elastic  sheaths  connected  with  elastic  fibres. 

Ranviei>has  used  the  same  membrane  for  studying  the  changes  which  take 
place  in  a striped  muscular  fibre  during  contraction. 


LESSON  XVIL 

NBRVB-FIBBBS, 

Nerve-fibres  are  of  two  kinds,  medullated  and  non-medullated ; 
the  former  are  found  chiefly  in  the  white  matter  of  the  nerve- 
centres  and  the  cerebro-spinal  nerves,  while  the  latter  occur  in 
large  numbers  in  the  sympathetic  system. 

I.  Medullated  Nerve-Fibre. — The  essential  part  is  the  axis- 
cylinder,  a soft,  transparent  rod  or  thread  running  from  end  to  end 
of  the  fibre,  and  composed  of  primitive  fibrils.  It  is  covered  by 
the  myelin,  or  white  substance  of  Schwann,  or  medullary  sheath, 
which  envelops  the  axis-cylinder  everywhere  except  at  the  termina- 
tion of  the  fibre  and  at  the  nodes  of  Ranvier  (fig.  181).  The 
myelin  gives  the  nerve-fibre  its  highly  refractive  appearance  and 
its  double  contour,  and  it  can  be  shown  to  consist  of  a stroma  or 
network  of  fibrils  of  a peculiar  chemical  substance  called  neuro- 
keratin, enclosing  a semifluid  fatty-like  substance,  containing, 
amongst  other  chemical  substances,  protagon,  a complex  phos- 
phorised  fat.  Histologically  it  consists  of  cylinder-cones  or  medul- 
lary segments,  whose  ends  are  bevelled  and  fit  one  into  the  other, 
but  separated  from  each  other  by  oblique  clefts  or  incisures. 

Outside  the  medullary  sheath  is  a thin,  transparent,  tough  elastic 
sheath,  the  primitive  sheath,  sheath  of  Schwann,  or  neurilemma. 
It  is  not  present  in  all  nerve-fibres,  being  absent  from  the  fibres  of 
the  central  nervous  system. 

Between  the  axis-cylinder  and  the  myelin  is  a thin  layer  of 
matter,  called  by  Kiihne  axilemma.  By  others  it  is  regarded  as 
an  albuminous  cement. 

At  fairly  regular  intervals — about  i mm. — along  the  course  of  a 
fibre  are  constrictions,  the  nodes  of  Ranvier,  where  the  myelin  is 
absent,  so  that  the  neurilemma  appears  to  produce  a constriction 
at  these  points.  The  part  between  any  two  successive  nodes  of 


XVII.] 


NERVE-FIBRES. 


203 


Ranvier  is  an  interannular  segment,  or 
internode,  and  about  the  centre  of  this, 
under  the  neurilemma,  is  a flattened  oval 
nucleus — nerve-corpuscle — surrounded  by 
a small  quantity  of  protoplasm,  and  lying 
in  a slight  depression  of  the  myelin. 
Nodes  of  Ranvier  are  absent  from  the 
nerve-fibres  of  the  brain  and  spinal  cord. 
Osniic  acid  blackens  the  myelin ; silver 
nitrate  produces  the  so-called  Ranvier’s 
crosses.  The  manner  of  their  production 
is  given  in  the  text.  Nerve-fibres  do  not, 
as  a rule,  branch  except  towards  their 
terminations. 

The  fibres  vary  greatly  in  diameter, 
some  being  only  half  as  broad  as  a red 
blood-corpuscle  (4  />t,  inch),  others  as 

broad  (8  /a,  3-2V0  i^ich),  and  others  broader 
still;  so  that  they  vary  in  diameter  from 

2 /A  to  20  fJL. 

II.  Non  - Medullated  Nerve -Fibres. — 

They  occur  specially  in  the  sympathetic 
system,  but  are  also  present  in  the  cerebro- 
spinal nerves.  Each  fibre  consists  of  a 
bundle  of  fibrils  enclosed  in  a transparent 
structureless  sheath.  Some  observers  doubt 
the  existence  of  this  sheath.  The  fibres 
are  somewhat  flattened ; they  branch  and 
anastomose,  and  in  their  course  are  oval 
nuclei  (fig.  182).  As  they  have  no  myelin, 
they  are  not  blackened  by  osmic  acid,  so 
that  this  reagent  serves  to  distinguish  the 
two  kinds  of  fibres. 

Nerve-trunks  consist  of  bundles  or  funi- 
culi of  nerve-fibres,  each  bundle  containing 
a greater  or  less  number  of  fibres.  Several 
bundles  are  held  together  by  a common 
connective-tissue  sheath — the  epineurium. 
The  sheath  around  each  funiculus  is  com- 
posed of  lamellated  connective  tissue, 
covered  on  both  surfaces  by  endothelial 
cells,  and  is  called  the  perineurium  or 
lamellated  sheath.  Lymph  spaces  exist 
between  the  lamellae.  Delicate  fibrils  of 
connective  tissue  lie  between  the  nerve- 


_ Node  of 
Ranvier. 


Primitive 

sheath. 


.Nerve 

corpuscle. 


- Axis-cylinder. 


, White  substance 
of  Schwann. 


Node  of 
"Ranvier. 


Fig.  181.  — Medullated  Nerve- 
Fibre.  Osmic  acid. 


204 


PRACTICAL  HISTOLOGY. 


[XVII. 


fibres,  and  constitute 
A 


the  endoneurium.  The  larger  blood-  and 
lymph-vessels  lie  in  the  epineurium  and 
perineurium,  while  the  endoneurium  sup 
ports  the  few  capillaries  which  are  distri- 
buted to  the  nerve-fibres  (fig.  190). 

The  term  Sheath  of  Henle  is  applied  to 
the  prolongation  of  the  perineurial  sheath — 
usually  a single  lamella  — around  a small 
branch,  or  even  one  or  two  nerve-fibres. 

The  following  statement  may  facilitate  the 
study  of  the  parts  to  be  investigated  : — 

Kerve-Fibres. 

I.  Medullated  (chiefly  in  cerebro-spinal 
system). 

II.  Non-Medullated  (sympathetic  or  fibres 
of  Kemak). 

A.  Medullated  Nerve  Fibre  consists  of — 

(i.)  Primitive  sheath,  sheath  of  Schwann 
(or  neurilemma). 

(2.)  Nerve-corpuscles  occur  under  the 
primitive  sheath  in  each  inter- 
annular  segment. 

(3.)  White  substance  of  Schwann,  myelin, 
or  medullary  sheath  (with  cylin- 
der cones  and  incisures).  It 
contains  a net-work  of  neuro- 
Jceratin. 

(4.)  Axis-cylinder,  composed  of  primi- 
tive fibrils  (surrounded  by  a 
sheath  called  the  axilemma). 

(5.)  Nodes  of  Ranvier  and  internodal  or 
ihterannular  segments  between 
two  successive  nodes. 

Ranvieds  crosses  and  Frommann^s  lines, 
obtained  by  using  nitrate  of  silver. 

Sheaths  of  a Nerve-Trunk. — Epineurium, 
perineurium,  and  endoneurium. 

B.  Non-Medullated  Nerve-Fibres  consist  of — 


Fig.  182. —Non-Medullated 
Nerve-Fibre,  Vagus  of  Dog. 
h.  Fibrils  ; n.  Nucleus  ; p. 
Protoplasm  surrounding  it. 


(i.)  A bundle  of  fibrils,  usually  enclosed  in 
(2.)  A transparent  sheath  or  neurilemma  (I), 
(3.)  On  the  fibres  are  oval  nuclei. 


XVII.] 


NERVE-FIBRES. 


205 


bm 


1.  Medullated  Nerve-Fibres  (H). — Select  a small  nerve,  e.g.^ 
the  sciatic  on  one  of  its  branches,  of  a frog.  Cut  out  half  an  inch 
of  it,  and  place  it  on  a dry  slide,  but  add  no  fluid.  Fix  one  end  of 
the  thread  by  pressing  on  it  with  any  thin  blunt  object,  e.g.,  the 
flat  surface  of  a mounted  needle,  and  fray  out  the  opposite  end  in 
a fan-shaped  manner  with  a mounted  needle,  so  as  to  isolate  some 
nerve-fibres.  To  prevent  it  from 
drying,  breathe  on  the  specimen 
from  time  to  time.  Add  a drop 
of  normal  saline,  cover,  and  ex- 
amine (flg.  183). 

(a.)  Observe  highly  refractive 
medullated  nerve:fibres  of  vari- 
able size,  some  as  broad  as  a red 
blood-corpuscle  and  others  nar- 
rower. Each  fibre  has  a double 
contour,  i.<\,  two  thin  lines  on 
each  side  of  the  centre.  The 
double  contour  may  be  inter- 
rupted here  and  there.  The 
double  contour  is  due  to  the 
white  substance  of  Schwann, 
medullary  sheath,  or  myelin. 

(&.)  In  the  centre  a clear 
bright  rod,  the  axis-cylinder. 

(c.)  Outside  the  myelin  is  a 
thin  transparent  sheath,  primi- 
tive sheath  or  neurilemma, 
scarcely  to  be  detected  as  such 
unless  tjie  fibre  is  ruptured  or 
the  sheath  raised  from  the 
myelin,  or  stretching  as  a funnel-shaped  prolongation  from  the  end 
of  a torn  fibre. 

{(I,)  Selecting  a fibre  isolated  for  a considerable  distance,  trace 
its  outline,  and  observe  at  intervals  slight  constrictions — nodes 
of  Ranvier — where  the  myelin  is  absent. 

(e.)  A small  quantity  of  delicate  connective  tissue — endoneurium 
— with,  perhaps,  a capillary  and  a few  blood-corpuscles  between  the 
fibres. 

(/.)  The  myelin  tends  to  exude  from  the  ruptured  ends  of  the 
fibres,  and  appears  as  highly  refractive  spherical  droplets — myelin 
drops — often  with  concentric  markings,  but  there  is  no  nucleus 
in  them  (fig.  183,  6).  The  myelin  drops  exude  more  rapidly  in 
a preparation  irrigated  with  i per  cent,  acetic  acid,  or  2 per  cent, 
caustic  soda,  or  even  with  distilled  water  alone. 


Fig. 


Fresh  FiG, 


Nerve -Fibre  Ex- 
amined in  Nor- 
mal Saline.  a. 
A short  piece  of 
the  axis-cylinder 
projecting  ; 6. 

Myelin  drops. 


184.  — Nerve- 
Fibre  of  Frog.  a. 
Ranvier’s  node ; h. 
Nucleus  ; i.  Inci- 
sures ; m.  Myelin 
blackened.  Osmic 
acid,  X 400. 


2o6 


PRACTICAL  HISTOLOGY. 


[XVII. 


Stain  the  preparation  with  picro-carmine,  and  note  that  this 
reagent  diffuses  into  the  fibres  at  their  cut  ends  and 
at  the  nodes  of  Kanvier.  It  stains  the  axis-cylinder 
red,  and  also  the  nerve-nuclei  or  corpuscles  which 
lie  just  under  the  neurilemma. 

2.  Nerve-Fibres  in  Osmic  Acid  (H).^ — A nerve- 
fibre  is  rapidly  blackened  by  osmic  acid,  as  can  be 
shown  by  applying  a drop  of  i per  cent,  solution  to 
a fresh  nerve,  but  for  good  permanent  preparations 
it  is  well  to  stain  the  nerve  after  the  action  of  the 
osmic  acid.  Place  a small  piece  of  nerve  in  a small 
glass  thimble  along  with  2 cc.  of  .5  per  cent,  osmic 
acid.  Cork  the  thimble,  and  after  twenty-four  hours 
thoroughly  wash  the  preparation  ; tease  it  a little, 
and  place  it  for  twenty-four  hours  in  a solution  of 
picro-carmine.  In  fact,  if  it  be  left  for  days  in  this 
dye  it  is  better,  as  the  fibres  can  then  be  more  readily 
dissociated.  Tease  a small  fragment,  and  mount  it 
in  glycerine  acidulated  with  formic  acid. 

(a.)  Observe  in  each  fibre  the  myelin  stained  black. 
Search  for  a node  of  Ranvier,  a narrow  constriction, 
and  note  that  the  myelin  is  absent  at  the  constriction, 
although  the  axis-cylinder  and  neurilemma  are  pre- 
sent (figs.  184,  a,  185)  Find  the  next  node,  and, 
between  the  two  adjoining  nodes,  the  stretch  of 
nerve — the  internodal  or  interannular  segment 
(fig.  1 81). 

(6.)  In  the  interannular  segment,  just  under  the 
neurilemma,  and  lying  in  a slight  depression  of  the 
myelin  (fig.  184,  6),  a red-stained  oval  nucleus  sur- 
rounded by  a small  quantity  of  protoplasm,  and 
about  midway  between  the  two  nodes  (fig.  185,  n). 
The  axis-cylinder  stained  red,  and  continuous 
throughout  the  fibre. 

(c.)  In  the  myelin  what  look  like  oblique  slits — 
incisures  — running  obliquely  outwards  from  the 
axis-cylinder  to  the  neurilemma  (fig.  185,  ^).  They 
correspond  on  each  side,  and  break  up  the  myelin 
^ Mbre^’ ^ number  of  short  lengths — cylinder  cones — 

Acid),  a.  Axis-  the  bevelled  end  of  one  cylinder-cone  fitting  into  the 
cvlinucr*  r IiOclG  • ^ 

of  Ranvier;  i 111- oppositely  bevelled  end  of  the  next  cylinder-cone. 

cieu^*  Many  cones  lie  in  an  internode. 

plasm’;  s.  Xeuri-  (^d.)  Some  of  the  fibres  are  broad,  and  others 
lemma.  narrow,  about  half  the  breadth  of  the  others. 

(e.)  Some  fibres  are  not  blackened  by  osmic  acid  at  all.  They 


P 

71 


XVII.] 


NERVE- FIBRES. 


207 


Fl0.»  186. — Nerve 
with  Ranvier’s 
Crosses.  Silver 
nitrate,  x 30. 


Nerve- 

Node 


appear  as  flattened  bands  with  oval  nuclei  at  intervals  in  their 
course.  They  are  non  medullated  nerve -fibres,  which,  as  they 
have  no  myelin,  are  not  blackened  by  the  osmic  acid. 

(/.)  A small  quantity  of  connective  tissue  and  capillaries.  To 
get  a good  view  of  the  incisures  and  nodes,  the  best  plan  is  to 
stretch  the  sciatic  or  other  nerve  of  a frog  on  a match  before 
placing  it  in  osmic  acid.  In  this  way  the  fibres  are  kept  straight, 
and  the  cylinder-cones  pulled  asunder  as  far  as  possible,  thus 
making  the  incisures  wide  and  distinct. 

3.  Ranvier’s  Crosses  (H). — Rapidly  tease  out  on  a dry  slide  one 
of  the  branches  of  the  sciatic  nerve  (frog),  and  stain  it  for  five 
minutes  with  .3  per  cent,  solution 
of  silver  nitrate.  Wash  off  the 
silver,  apply  a drop  of  glycerine, 
and  expose  it  to  daylight.  It 
rapidly  becomes  brown. 

(a.)  Observe  the  fibres,  but  at 
each  node  will  be  seen  a brownish- 
black  cross  (fig.  186);  the  silver 
nitrate  diffuses  into  the  nerve-fibre 
only  at  the  nodes,  stains  the 
cement  joining  one  internode  with 
another,  thus  making  the  trans- 
verse bar  of  the  cross,  and  as  it 
diffuses  along  the  axis-cylinder  it 
stains  some  cement  substance  on 
the  latter,  and  thus  makes  the 

vertical  bar  of  the  cross.  Occasionally  a number  of  transverse 
(fig.  187)  lines — Frommann’s  lines — are  seen  on  the  axis-cylinder, 
^.e.,  on  the  vertical  bar  of  the  cross  (p.  210).  Place  a sciatic  nerve 
of  a frog  for  twelve  hours  in  i per  cent.  AgNO^,  and  keep  it  in 
the  dark.  Wash  and  harden  in  absolute  alcohol,  and  mount 
in  balsam.  The  crosses  and  lines  are  then  seen  with  great  dis- 
tinctness. 

4.  Endothelial  Cells  of  the  Perineurium.  Intercostal,  or  other 
Small  Nerve.— (i.)  It  is  well  also  to  stain  with  silver  nitrate  one 
of  the  small  intercostal  nerves  of  a rat  or  some  other  small  nerve. 
Stain  the  whole  nerve  in  silver  nitrate. 

(ii.)  Open  the  abdomen  of  a frog,  remove  the  abdominal  viscera, 
so  as  to  expose  the  nerves  coming  from  the  vertebral  canal.  Pour 
on  the  nerves  .3  per  cent,  silver  nitrate.  After  three  minutes,  cut 
out  the  nerves  and  place  them  for  half  an  hour  in  fresh  .3  per 
cent.  AgNOg.  Wash  in  distilled  water,  tease  a piece  in  glycerine, 
and  expose  it  to  light. 

(a.)  The  crosses  are  seen  as  before,  but  above  them  is  the 


Fig.  187.  - 
Fibre.  ( 
and  cross  of  Ran- 
vier  with  Fro- 
mann's  lines,  x 
200.  Silver  nit- 

rate. 


PRACTICAL  HISTOLOGY. 


208 


[XVII. 


endothelial  sheath  of  the  nerve-fibre,  composed  of  polygonal 
squames  (fig.  189). 

5.  Axis-Cylinder. — Harden  for  two  or  tliree  days  a nerve  in 
chromate  of  potash,  tease  a piece,  and  stain  it  in  carmine.  Observe 
the  axis-cylinder  stained  red  (fig.  188).  Or  treat  a fine  nerve  for 
four  days  to  a week  in  per  cent,  chromic  acid  or  ^ per  cent. 

bicliromate  of  potash.  On  teasing  long  stretches  of 
isolated  nerve  axis-cylinders  are  readily  found. 

6.  T.S.  of  a Nerve. — Select  a rather  large  nerve, 
e.g.y  the  liuman  sciatic,  and  harden  about  an  inch  of 
it  in  picric  acid  for  forty-eight  hours,  or  in  2 per 


cylinder;  b.  Ran- 
vier’s  node  ; c. 
Nucleus.  Chro- 
mate of  potash 
and  carmine,  x 
200. 


cent,  ammonium  bichromate  for  two  weeks, 
out  the  bichromate. 

Complete  the  harden- 
ing in  alcohol.  Sec- 
tions may  be  made,  and 
stained  with  logwood 
or  carmine,  but  they 
are  very  apt  to  fall  to 
pieces.  It  is  prefer- 
able, therefore,  to  stain 
the  hardened  tissue 
“ in  bulk.”  • Place  it 
in  borax-carmine  for 
three  days,  then  trans- 
fer it  to  acid  alcohol, 
and  pass  it  through 
absolute  alcohol  and 
turpentine,  and  embed 
it  in  paraffin.  Cut 
sections,  when  the 
paraffin  keeps  all  the 
])arts  in  their  places. 

Fix  a section  on  a slide  with  white  of  egg. 


Wash 


Fig.  189.  — Intercostal  Nerve  of 
Mouse,  Ranvier’s  Crosses  and 
Endothelial  Covering.  AgNOs, 
X 300. 


remove 

all  the  paraffin  by  placing  the  slide  in  turpentine, 
clear  it  up  in  clove-oil,  and  mount  in  balsam. 

("•)  (L)  Observe  the  connective-tissue  sheath — 
epineurium — or  sheath  surrounding  the  whole 
nerve,  sending  processes  into  the  nerve, — numerous 
bundles — some  large,  others  smaller — or  funiculi  of  nerve-fibres,  each 
surrounded  by  a lamellated  sheath — perineurium — which  sends  fine 
septa — endoneurium — into  each  funiculus.  In  each  bundle  the  cut 
ends  of  the  fibres  are  directed  towards  the  observer  (fig.  190). 

The  large  blood-vessels  are  in  the  epineurium,  and  a few  in  the 
endoneurium. 


XVII.  ] 


NERVE-FIBRES. 


209 


(b.)  (H)  Select  a bundle.  Observe  the  perineurium^  made  up 
of  several  concentric  lamellae  with  nuclei  between  them.  The  cut 
ends  of  the  fibres  varying 
in  diameter. 

(c.)  Xote  in  each  fibre 
the  section  of  the  stained 
axis-cylinder.  Surround- 
ing this  a clear  trans- 
parent ring,  indicating 
the  position  of  the  myelin, 
which  has  been  dissolved 
out  in  the  process  of  pre- 
paration. 

(cZ.)  Outside  this  a thin 
circle  — the  primitive 
sheath.  Between  the 
nerve-fibres  a small 
quantity  of  connective 
tissue  or  endoneurium. 

Non-Medullated  Nerve- 
Fibres. 


Fig.  190.— T.S.  of  Several  Funiculi  of  the  Median 
Nerve,  p.  Perineurium  ; ep.  Epineurium ; ed.  En- 
doneurium. 


7.  Non-Medullated  or 
Sympathetic  Nerve  - 

Fibres. — (i.)  These  are  readily  found  in  the  large  splenic  nerve  of 
;he  ox,  or  in  a portion  of  the  sympathetic  nerve  of  the  thoracic  or 
abdominal  chain.  The  nerve  has  a pretty  thick  sheath.  Cut  this 
open,  and  cut  off  a small  piece.  Tease  it  in 
normal  saline.  The  process  of  teasing  is 
greatly  facilitated  by  placing  the  nerve  for 
twenty-four  hours  in  dilute  acetic  acid  (5  drops 
in  100  cc.  water). 

(H)  Observe  that  there  are  very  few  medul- 
lated  fibres,  the  great  majority  being  non- 
medullated  (fig.  192).  Their  outlines  are  not 
very  distinct ; they  are  faintly  striated  longi- 
tudinally, and  have  oval  nuclei  at  intervals. 

They  may  be  stained  with  picro-carmine. 

(ii.)  Place  the  cervical  sympathetic  nerve  of 
a rabbit  in  .25  per  cent,  osmic  acid  for 
twenty-four  hours.  Wash  it  in  water,  and  stain  it  for  several 
hours  in  picro-carmine.  Tease  a fragment  in  glycerine. 

(iii.)  Or  tease  the  vagus  of  a rabbit  upon  a dry  slide,  taking  care 
that  the  nerve  does  not  become  dry.  Cover  it  for  5-10  minutes 

O 


Fig.  191.— Non-Medullater 
Nerve  - Fibres,  Sympa- 
thetic Nerve  of  Rabbit, 
X 200. 


210 


PRACTICAL  HISTOLOGY. 


[XVII 


with  I per  cent,  osmic  acid,  wash  away  the  osmic  acid,  add  picro- 
car’mine,  and  place  the  whole  for  24-48  hours  in  a moist  chamber 


Osmic  Acid.  There  are  two  medullated  fibres  in  a Medullated 

amongst  Remak’s  fibres,  x 350,  Externally  the  Nerve-Fibre. 

epineurium,  Epn;  mF.  Medullated  fibres  with 

Ranvier’s  nodes;  K.  Nuclei  of  Remak's  fibres; 

mlF.  Remak’s  fibres ; K'.  Nucleus  of  medullated 

fibre  ; Bz.  Connective-tissue  cells. 


(fig.  47),  and  afterwards  displace  the  picro-carmine  by  glycerine, 
as  recommended  at  p.  82. 

(H)  Observe  a few  small  medullated  fibres  (blackened  by  OsO^), 
and  numerous  non-medullated  fibres,  some  of  which  may  be  seen  to 
branch.  Xote  the  oval  nuclei  on  the  fibres  (fig.  191).  the  part 
be  taken  from  near  a ganglion,  often  nerve-cells  may  be  seen. 


ADDITIONAL  EXERCISES. 

8.  Neurokeratin  Network  and  Axis-Cylinder. — Place  the  fresh  sciatic 
nerve  of  a frog  in  a dilute  solution  of  ferric  chloride  consisting  of — 

Liquor  ferri  perchloridi  . . . . i part 

Distilled  water  or  spirit  . . . *3-4  parts. 


XVII.] 


NERVE-FIBRES. 


2II 


Fig. 


i. — From- 


niann’s  Lines 
on  an  Axial 
Cylinder. 


Leave  it  in  this  fluid  for  three  or  four  days.  Wash  every  trace  of  the  iron  salt 
out  of  the  preparation,  and  preserve  it  in  alcohol.  Place  small  pieces  of  the  nerve 
several  days  in  a saturated  solution  of  dinitrosoresorciii  in  75  per  cent,  alcohol. 
Tease  a fragment,  dehydrate  it  with  alcohol,  clarify  with  xylol, 
and  mount  in  balsam  [Platner).  This  is  an  excellent  method. 

{a.)  Observe  the  axis-cylinder  stained  green.  It  can  be 
seen  with  the  utmost  distinctness  passing  from  one  internode 
to  the  next  one,  and  across  the  nodes  of  Ranvier,  which  are 
particularly  sharply  defined.  In  the  myelin,  a network  of 
fibres — the  neurokeratin  network — stained  green.  The  axis- 
cylinder  appears  as  distinct  as  in  fig.  193. 

{h.)  Klihne  and  Ewald’s  Method.^ — Harden  a nerve  for 
twenty-four  hours  in  absolute  alcohol.  Boil  it  in  absolute 
alcohol,  and  extract  it  with  ether.  To  remove  everything 
except  the  network,  a teased  preparation  is  digested  in  pan- 
creatic juice. 

(c.)  Heidenhain’s  luematoxylin  (p.  70)  may  be  used  to 
stain  nerve-fibres.  It  stains  the  axis-cylinder  and  the  neuro- 
kcratin  network. 

9.  Frommann’s  Lines  and  Ranvier’s  Crosses  (H). — Place 
a fresh  nerve  in  .5-1  per  cent,  silver  nitrate  for  forty-eight 
hours,  and  keep  it  in  the  dark.  Wash  it  in  water,  and 
expose  it  to  light  for  2-3  days  in  equal  ])arts  of  formic  acid, 
water,  and  glycerine,  and  preserve  it  in  glycerine.  Tease 
a piece  in  glycerine. 

(a.)  Observe  the  crosses  of  Ranvier  .sharply  defined,  and  on  the  axis-cylinder 
well-defined  transverse  markings,  extending  for  a long 
distance  along  the  axis-cylinder.  Frommann’s  lines. — If 
an  axis-cylinder  be  dislodged  from  its  fibre,  a hiconical 
swelling  may  be  seen  It  corresponds  to  that  part  of  the 

axis-cylinder  opposite  a node  of  Ranvier  (fig.  194). 

10.  Axis-Cylinder — («.)  Action  of  Collodion  (H). — Tease 
a fresh  sciatic  nerve  of  a frog  without  adding  any  fluid. 

Add  a large  drop  of  collodion  and  apply  a cover-glass,  or 
tease  a fresh  nerve  in  chloroform.  Examine  quickly,  as  the 
preparation  soon  spoils.  The  neurilemma  is  distinct,  the 
myelin  is  transparent  and  finely  granular,  while  the  axis- 
cylinder  appears  as  a dark  cylindrical  rod — often  with  a 
curved  course — in  the  centre  of  the  fibre,  and  it  may  even 
project  beyond  the  end  of  the  fibre. 

(h.)  Isolated  axis-cylinders  are  readily  obtained  from  the 
central  nervous  system  after  maceration  of  the  white  matter 
of  the  cord  in  methyl-mixture  (p.  26),  or  Muller’s  fluid  or 
ammonium  chromate. 

11.  Schwann’s  Sheath. — Macerate  a peripheral  nerve  for 
several  days  in  ammonium  chromate  (i  : 3000).  The  myelin 
is  dissolved  wdiile  the  neurilemma  and  axis-cylinder  remain 
( Schieffe  r decker) . 

12.  T.S.  Nerve,  Osmic  Acid  (H). — Stretch  a nerve  on  a 
piece  of  wood,  and  ]dace  it — wood  and  all — for  two  days  in 
.5  per  cent,  osmic  acid,  or,  better  still,  in  Flemming’s 
mixture  for  one  day.  On  the  second  day,  add  a little  more 
osmic  acid  to  Flemming’s  mixture,  and  harden  the  nerve 
for  another  day.  It  is  better  to  embed  the  nerve  in  paraffin 
and  make  transverse  sections.  The  sections  are  fixed  on  a slide  by  a fixative, 

^ Kuhne's  UTitersuch.^  Heidelberg,  1878. 


Fig.  19:;.— Periphe- 
ral Nerve-Fibre 
of  Frog.  a.  Lon- 
gitudinal fibrilla) 
in  axis-cylinder; 
h.  T.S.  of  nerve- 
fibre.  Osmic  acid 
and  Bismarck 
brown,  x 1000. 


212 


PRACTICAL  HISTOLOGY, 


[XVII. 


the  paraffin  is  extracted  by  turpentine,  and  the  sections  mounted  in  balsam. 
They  may  be  stained  with  a watery  solution  of  Bismarck  brown.  Instead  of 
this,  after  fixing  the  nerve  in  osmic  acid,  nnd  hardening  in  90  per  cent.,  alcohol, 
])lace  it  for  24-48  hours  in  a strong  saturated  watery  solution  of  acid  fuchsin 
(S.N.  30),  wash  in  alcohol,  embed  and  cut  in  paraffin.  The  ends  of  the  fibrils 
on  the  axis-cylinder  are  stained  red  (fig.  195). 

(a.)  Note  the  axis-cylinder  in  the  centre  surrounded  by  a dark  ring  (figs. 
195,  196,  J),  the  myelin  blackened  by  the  OSO4.  If  the  section  of  a nerve- 
fibre  is  through  incisures,  the  double  contour  of  the  myelin  may  be  seen 
sometimes  with  a narrow  black  edge,  at  others  with  a broad  black  edge 
externally. 

(b.)  If  a piece  of  the  nerve  be  placed  for  forty-eight  hours  in  a solution  of 
Bismarck  brown,  and  then  teased,  the  appearance  shown  in  fig.  195,  a,  is 
obtained,  when  the  axis-cylinder  presents  a longitudinally  striated  appearance. 

13.  Size  of  Nerve-Fibres. — The  osmic  acid  method  has  yielded  the  best 
results.  Some  of  the  fibres  are  broad,  and  others  are  narrow  or  fine.  Thus  the 
anterior  roots  of  the  upper  cervical  nerves,  and  the  third  cranial  nerv'e,  contain 
only  broad  nerve-fibres,  while  the  second  and  succeeding  thoracic  nerves  contain 
broad  and  fine  fibres  (fig.  196).  In  nerves  going  to  muscles  there  are  many 
large  and  few  small  medullated  fibres,  while  in  nerves  going  to  viscera  the 
fine  medullated  fibres  are  far  more  abundant  than  the  broad  fibres. 


Fig.  196. — A.  T.S.  of  the  anterior  root  of  a spinal  nerve  below  the  first  dorsal  nerve. 
B.  T.S.  of  a part  of  a cervical  nerve.  Osmic  acid. 


14.  Living  Nerve-Fibres  are  readily  studied  in  the  inflated  lungs  of  a newt 
or  frog.  The  frog’s  lung  is  best  kept  inflated  by  Holmgren’s  apparatus.^ 
Nerve-fibres  are  also  readily  seen  in  the  tongue  of  a frog  arranged  as  for 
studying  the  circulation  of  the  blood  (Lesson  XIX.). 

15.  Marchi’s  Method  for  Degenerated  Fibres. — Harden  a nerve  in  Muller’s 
fluid  for  eight  days  and  then  in  the  following  fluid  : — 


Embed  in  celloidin  and  mount  in  warmed  balsam.  It  is  well  not  to  employ 
balsam  dissolved  in  chloroform,  as  then  the  darkdied  parts  lose  their  dark 
colour.  The  degenerated  parts  of  nerve-fibres  are  black.  This  method  is 
particularly  useful  for  degenerations  in  the  nerve-centres  before  sclerosis  has 

^ Beitrdge  z.  Anat.  u.  Phys.^  C,  Ludwig^  gewidmet^  Leipzig,  1874,  p.  cxvi. 


Marclii's  Fluid. 


^Hiller’s  fluid 

Osmic  acid  (i  per  cent)  . 


2 parts. 
I part. 


XVII.] 


NERVE-FIBRES. 


213 


set  in,  but  it  is  also  applicable  to  nerve-fibres  that  have  underi^one  degenera- 
tion, e.g.^  after  section  of  a nerve,  constituting  Wallerian  degeneration.  It 
will  also  detect  any  degenerated  fibres  in  an  ordinary  nerve. 

16.  Isolated  Schwann’s  Sheath. — Place  a stretched  nerve  of  a frog  in  the 
following  fluid  for  tw'enty-four  hours  in  the  dark  : — 

Boveri’s  Fluid 

Silver  nitrate  ( I per  cent.)  . . . 10  cc. 

Osinic  acid  (i  percent.)  . . . 10  ?? 

Wash  it  in  water,  and  place  for  twenty-four  hours  in  very  dilute  caustic 
potash  (2-3  drops  of  a concentrated  solution  in  15  cc.  water).  Tease  in 
glycerine.  The  axis-cylinder  shrinks  and  Schwann’s  sheath  may  be  traced  as 
a continuous  sheath  without  any  interruptions  at  the  nodes. 

17.  Nerve-Fibres  of  the  Spinal  Cord. — These  are  devoid  of  Schwann’s 
sheath,  but  they  possess  both  Ranvier’s  nodes  and  incisures  of  Lantermann. 
Boveri’s  fluid  may  stain  both.  It  is  evident  then  that  these  two  structures 
have  no  relation  to  Schw^ann’s  sheath,  but  are  related  entirely  to  the  myelin. 
The  cylinder- cones  are  readily  isolated  by  Schiefferdecker’s  methyl-mixture 
(p.  26). 

18.  Degeneration  of  Nerve-Fibres. — This  is  readily  studied  in  the  rabbit. 
The  skin  is  first  disinfected  with  solution  of  corrosive  sublimate,  and  then  the 
median  and  ulnar  nerves  are  exposed  on  the  inner  aspect  of  the  upper  arm, 
the  nerves  divided,  and  the  wound  sealed  with  collodion  (C.  Huber). ^ In 
different  animals  the  nerves  are  excised  2,  3,  4,  . . . 8 or  10  days  after  the 
operation.  The  excised  nerves  are  kept  extended  on  wood  and  fixed  for 
twenty-four  hours  either  in  Hermann’s  fluid  (Lesson  XXXV.)  or  the  picro- 
osmium  mixture  of  Benda,  prepared  by  saturating  a i per  cent,  solution  of 
osniic  acid  with  picric  acid  and  filtering.  They  are  then  washed  in  \vater  and 
hardened  in  alcohol.  The  sections  are  stained  with  safranin  and  light-green. 
Besides  showing  the  usual  degeneration  phenomena,  they  show  mitotic  division 
of  the  nuclei  of  Schwann’s  sheath,  showing  that  these  proliferate. 


LESSON  XYIII. 

NERVE -GANGLIA,  NERVE -CELLS,  AND  PERI- 
PHERAL TERMINATIONS  OP  MOTOR  NERVES. 

Spinal  Ganglia. — Harden  a spinal  ganglion  of  a cat  or  dog  in 
2 per  cent,  ammonium  bichromate  for  three  weeks,  and  subse- 
quently in  alcohol.  Make  transverse  and  longitudinal  sections 
of  the  ganglion,  stain  with  logwood  or  carmine,  and  mount  in 
balsam. 

1.  L.S.  Mammalian  Spinal  Ganglion  (L). — (a.)  Note  the  cap- 
^ ArcMv  f.  mik,  Aiiat.y  xl.  p.  409,  1892. 


214 


PRACTICAL  HISTOLOGY. 


[XVIII. 

sule  (fig.  197,  c)  surrounding  the  ganglion;  nerve-fibres  (a)  enter 
the  ganglion  at  one  end  and  leave  it  at  the  other.  They  run  in 

groups,  chiefiy  through  the  central  part  of  the  ganglion,  so 
that  they  are  cut  in  different  planes. 

(6.)  JN’umerous  spherical  cells  (fig.  197,  b)  lying  singly  or  in 
groups  between  the  nerve-fibres,  but  chiefly  towards  the  surface. 

(c.)  (H)  Select  a single  ganglion-cell;  note  its  spherical  shape, 


its  granular  contents,  and  single,  large,  distinct,  excentrically-placed 
nucleus,  often  with  one  or  more  distinct  nucleoli  (fig.  198).  The 
nucleus  has  a well-defined  nuclear  membrane. 

(d.)  Around  each  cell  is  a capsule,  which  is  lined  by  a single 
layer  of  flattened  cells,  but  only  the  nuclei  of  these  cells  are  seen. 

The  cell-substance  is  frequently  somewhat  re- 
tracted from  the  capsule,  so  that  a space  may 
intervene  between  the  two. 

In  a T.S.  of  such  a ganglion,  notice  the  capsule 
of  the  ganglion  sending  in  coarse  septa,  the  nerve- 
cells  near  the  circumference,  and  the  nerve-fibres 
chiefly  in  the  centre. 

2.  Isolated  Cells  of  a Spinal  Ganglion 
(Mammal)  (H). — Into  a dorsal  ganglion  of  a 
young  rabbit  make  an  interstitial  injection  of 
osmic  acid  (2  per  cent.).  Tease  a small  piece  in 
picro-carmine  and  mount  the  preparation  in 
glycerine.  Sometimes  a cell  with  its  single 
process  may  be  found.  The  cells  are  unipolar.  It  is  more  difficult 
to  find  the  connection  of  the  issuing  axis-cylinder  with  a nerve- 
fibre,  forming  what  Kanvier  has  described  as  T-shaped  nerve-fibres, 
but  with  care  such  processes  can  be  found. 


Fm.  198.— Two  Cells  in 
a Spinal  1 Ganglion 
(Human),  the  Proto- 
plasm shrunk  from 
the  Capsule,  x 200. 


XVIII.] 


NERVE-GANGLIA,  NERVE-CELLS,  ETC. 


215 


3.  Spinal  Ganglion  of  Frog. — These  ganglia  lie  under  cover  of 
the  small  white  calcareous  sacs  situated  on  each  side  of  the  verte- 
bral column,  which  are  seen  at  once  when  thfe  abdominal  cavity 
is  opened  and  the  abdominal  viscera  removed.  Kemove  the  white 
chalky  mass,  and  the  greyish  semi-transparent  small  ganglion  will 
be  seen.  With  sharp-pointed  forceps  it  is  not  difficult  to  tear  away 
the  capsule  of  the  white  calcareous  mass.  These  sacs  contain  arra- 
gonite ; some  of  the  crystals  are  large,  but  the  smaller  arragonite 
particles  when  examined  in  water  exhibit  Brownian  movement. 
Treat  it  in  the  same  way  as  directed  for  the  frog’s  Gasserian 
ganglion. 

(H)  In  a carefully-teased  specimen  (use  a dissecting  microscope, 
p.  22),  it  is  by  no  means  difficult  to  find  large  unipolar  cells,  each 
cell  with  a distinct  hyaline  capsule,  and  the  cell  itself  with  a 
relatively  large  nucleus  and  well-defined  nucleolus.  Moreover,  the 
continuation  of  the  body  of  the  cell  with  a nerve-fibre  is  not 
difficult  to  establish.  The  methylene-blue  method  may  be  used 
(p.  222). 

4.  Spinal  Ganglion  of  a Skate. — Make  an  interstitial  injection 
of  osmic  acid  (2  per  cent.)  into  such  a ganglion.  Stain  a piece  in 
picro-carmine  and  tease  it  in  glycerine.  Bipolar  cells  are  readily 
found.  Each  cell  shows  a distinct  capsule  enclosing  a nucleated 
cell  with  a pole  at  either  end 
continuous  with  a nerve-fibre 
(fig.  199). 

5.  G8.SS6riftll  Ganglion.  fig.  199. — Bipolar  Ganglion  Cell  of  the  Spinal 

{a.)  The  Gasserian  ganglion  Ganglion  of  a Skate. 

of  a sheep  does  very  well ; 

harden  it  in  the  same  way  as  for  spinal  ganglia  or  in  Muller’s  fluid. 
The  same  general  arrangement  of  fibres  and  cells  is  seen,  only  the 
cells  are  larger,  and  their  protoplasm  frequently  contains  granules 
of  a yellow  pigment.  Very  instructive  results  are  obtained  by 
double-staining  it  with  eosin  and  haematoxylin,  first  with  haemato- 
xylin  and  then  with  eosin,  or  use  eosin-haematoxylin,  and  mount 
in  balsam.  The  nuclei  are  blue,  the  other  parts  reddish.  If  a cell 
be  isolated  after  interstitial  injection  of  osmic  acid,  as  recom- 
mended for  spinal  ganglia,  the  cells  have  the  form  shown  in 
fig.  200. 

(6.)  A fresh  ganglion  may  be  teased  in  salt  solution.  The  large 
spherical  cells  are  readily  isolated,  but  they  usually  shell  out  of  their 
capsule.  Stain  them  with  magenta  solution. 

6.  Gasserian  Ganglion  of  Frog. — Destroy  the  brain  and  spinal 
cord  of  a frog,  remove  the  lower  jaw,  divide  the  skull  into  two  longi- 
tudinally by  a vertical  incision.  Tear  off  the  mucous  membrane 
covering  the  roof  of  the  mouth.  From  a foramen  just  behind  the 


2t6 


PRACTICAL  HISTOLOGY. 


[XVIII. 


eyeball  there  issue  a few  fine  threads,  branches  of  the  fifth  nerve. 
Scoop  out  the  brain.  These  threads  are  readily  recognised  by  being 
usually  somewhat  pigmented.  With  a pair  of  scissors  make  a snip 
in  the  base  of  the  skull  at  right  angles  to  the  cut 
already  made.  Turn  up  the  bone,  and  on  the  fifth 
nerve,  which  runs  towards  the  foramen  behind 
the  eyeball,  will  be  found  a small  oval  swelling 
surrounded  by  a tough  capsule.  Divide  the  latter 
and  remove  the  ganglion.  I have  usually  found 
that  the  little  ganglionic  swelling  is  somewhat 
pigmented.  At  any  rate,  it  is  easily  found  by 
tracing  the  fifth  nerve  backwards.  The  nerve  is 
accompanied  by  an  artery. 

Tease  the  ganglion  in  .25  per  cent,  osmic  acid, 
and  let  it  stain  in  this  fluid  for  two  hours.  Stain 
it  for  several  hours  in  picro-carmine  (under  a 
moist- chamber,  p.  82),  tease  a fragment  in 
glycerine.  Try  to  find  a cell  with  its  single  pro- 
cess prolonged  into  a nerve-fibre.  In  most  of  the 
cells,  however,  the  process  is  apt  to  be  detached. 

7.  Sympathetic  Ganglia  (Frog). — Lying  in 
contact  with  the  spinal  column  of  the  frog  is  a 
row  of  small  semi-transparent  ganglia,  the  sym- 
pathetic chain,  and  between  them  and  the  roots 
of  the  spinal  nerves  pass  fine  nerve  filaments. 
Open  the  abdomen  of  a freshly-killed  frog,  re- 
move the  intestinal  tract  and  liver,  cut  through 
the  peritoneum  above  the  kidneys,  raise  the 
kidneys  and  excise  them.  There  will  be  seen 
the  white  nerves  issuing  from  the  cord.  Between 
these  and  the  sympathetic  ganglia  fine  nerves  run  transversely. 
Cut  out  the  sympathetic  ganglia  and  treat  them  with  chloride  of 
gold  by  the  method  (p.  79,  3) ; or  cut  out  the  aorta  and  the 
adjacent  tissues,  and  subject  them  to  the  gold  chloride  method. 
After  the  piece  of  tissue  has  acquired  a purplish  colour,  examine 
it  with  a low  power  to  find  nerve-cells.  The  nerve-cells  may  be 
isolated  or  arranged  in  groups.  It  requires  great  care  to  get  a satis- 
factory preparation.  Note  the  pyriform  shape  of  the  cell,  each  with 
a large  nucleus,  the  cell-substance  continued  into  a straight  process, 
which  may  be  seen  to  be  encircled  by  a spiral  process.  The  body 
of  the  cell  is  surrounded  by  a well-marked  capsule,  which  is  con- 
tinued over  the  cell-processes,  and  has  nuclei  on  its  inner  surface. 

8.  S5nnpathetic  Ganglion  (Mammal)  (H). — Harden  the  first 
thoracic  sympathetic  ganglion  or  the  superior  cervical  ganglion  of  a 
man  or  rabbit  in  2 per  cent,  ammonium  bichromate  (2-3  weeks), 


Fig.  200.— Nerve-Cell 
from  Rabbit’s 
Spinal  Ganglion. 
X.  Nuclei  of  the 
cell  - capsule  ; n. 
Nucleus  of  nerve- 
fibre  ; a.  Nerve- 
fibre  ; Fibre  di- 
viding at  e at  a 
node  of  Ranvier, 
T-shaped  fibre. 


XVIII.] 


NERVE-GANGLIA,  NERVE-CELLS,  ETC. 


217 


Fig.  201.— Human  Superior  Cervical  Sym- 
pathetic Ganglion.  A.  Small  artery  ; 
C Capillary ; V.  Vein ; K.  Capsule ; 
N.  Nerve-cell,  x 300. 


and  subsequently  in  alcohol.  Make  transverse  sections.  Stain  in 
picro-carniine  or  a watery  solution  of  nigrosin  (several  hours),  and 
mount  the  former  in  Tarrant’s  solution,  and  the  latter  in  balsam. 

(a.)  Observe  the  fibrous  capsule  of  the  ganglion  ending  in 
septa,  and  numerous  bundles  of  non-medullated  nerve-fibres  cut 
obliquely  or  transversely.  A few  blood-vessels. 

(h.)  The  nerve-cells,  each  with  a capsule,  showing  nuclei.  The 
nucleated  cell  substance  is  frequently  somewhat  shrunk  from  its 
capsule,  and  at  one  side  it  usually 
contains  some  yellowish-brown  i)ig- 
ment  granules,  especially  if  the 
human  cervical  ganglion  be  used. 

It  is  difficult  to  see  the  process, 
which  becomes  continuous  with 
a nerve-fibre,  but  with  care  it  may 
be  seen  passing  out  of  one  or  more 
of  the  cells  (fig.  201). 

(c.)  The  veins  have  long  fusiform 
dilatations  upon  them ; this  is  not 
unfrequently  seen  in  teased  prepara- 
tions of  a human  ganglion,  but  can 
only  be  fully  demonstrated  in  an 
injected  specimen  of  the  ganglion. 

9.  Isolated  Multipolar  Nerve-Cells  of  the  Spinal  Cord. — 

There  are  several  ways  of  }>reparing  these. 

(i.)  Cut  out  a small  part  of  the  anterior  cornu  of  the  spinal 
cord  of  an  ox,  calf,  sheep,  or  other  animal,  and  place  it  in  very 
dilute  chromic  acid  (.01  per  cent.)  or  .2  per  cent,  potassium 
bichromate  for  a few  days,  and  do  not  change  the  fluid.  Wash, 
and  place  it  for  twenty-four  hours  in  strong  carmine  solution 
(p.  63).  Place  a little  of  the  red  pulp  on  a slide,  and,  with  the 
aid  of  a dissecting  microscope,  try  to  isolate  one  or  more  multi- 
polar nerve-cells. 

(ii.)  Or,  what  is  a better  method,  take  small  fragments  of  the 
anterior  cornu  of  the  spinal  cord  of  an  ox  or  calf,  and  place  them 
in  dilute  alcohol  for  forty-eight  hours  or  longer.  After  this  time 
we  can  see  better  the  distinction  between  the  grey  and  the  white 
matter.  Shake  the  fragments  in  the  dilute  alcohol,  and  allow  the 
debris  to  subside.  Pour  off  the  alcohol,  and  ‘‘  fix  ” the  cells  with 
.25  per  cent,  osmic  acid  (one  hour);  pour  this  off,  and  stain  the 
cells  for  forty-eight  hours  with  picro-carmine.  Pour  off  the  picro- 
carmine  and  replace  it  by  glycerine-jelly.  When  the  glycerine- 
jelly  is  warmed,  a drop  of  the  fluid  placed  on  a slide  is  almost 
certain  to  contain  one  or  more  isolated  multipolar  nerve-cells. 

With  a low  power  find  a cell. 

20 


2I8 


PRACTICAL  HISTOLOGY. 


[XVIII. 


(a.  ) (H)  Observe  the  large  size  of  the  cell  (loo  /x,  inch,  and 
therefore  visible  to  the  naked  eye),  with  numerous  processes — 
branched  processes — which  run  in  all  directions  (fig.  202).  The 

processes  branch ; and  then 
branch  again  and  again  to 
form  a fine  protoplasmic 
system  of  processes  — the 
protoplasmic  processes. 
One  process  is  always  un- 
branched, it  is  by  no 
means  difficult  to  see — the 
axis-cylinder  process  — 
which  becomes  continuous 
with,  or  in  fact  is,  or  be- 
comes, the  axis-cylinder  of 
a nerve-fibre  (fig.  202,  a). 

(6.)  The  multipolar  cell 
itself  has  no  cell- wall,  and 
it  contains  a large,  con- 
spicuous, spherical,  nucleo- 
lated  nucleus,  the  latter 
with  a distinct  envelope. 
The  protoplasm  is  fibril- 

terior  Cornu  of  the  Grey  Matter  of  the  Human  lofprl  onrl  tV>P  GHrilG 
Spinal  Cord.  a.  Axis-cylinder  process ; ft.  Pig-  nmilS  may 

ment,  x 150.  be  seen  to  stretch  into  the 

branched  processes.  Some- 
times the  cells  contain  pigment  (fig.  202,  h). 

10.  Cover-Glass  Preparation  of  multipolar  Nerve-Cells. — 
(i.)  From  a perfectly  fresh  cord  of  a sheep  or  ox  snip  off  a small  piece 
of  the  anterior  cornu;  press  it  between  two  cover-glasses,  so  as  to  form 
a thin  film.  Separate  the  cover-glasses  and  allow  the  film  adhering 
to  each  to  dry.  Float  the  cover-glass — film  surface  downwards— 
on  a concentrated  watery  solution  of  methylene-blue  for  several 
hours.  Wash  the  cover-glass  in  water  mixed  with  alcohol,  drain  it, 
allow  it  to  dry,  and  mount  it  in  xylol  balsam  ; the  cover-glass  can 
be  passed  through  absolute  alcohol,  cleared  with  xylol,  and  mounted 
in  xylol  balsam.  The  multipolar  nerve-cells  are  all  deeply  stained 
blue  {Thankoffer). 

(ii.)  If  the  use  of  aniline  colours  be  objected  to,  the  following 
method  gives  good  results  : — For  three  or  four  days  macerate  a small 
part  of  the  grey  matter  of  the  anterior  cornu  in  20  cc.  of  water, 
containing  i gram  of  each  of  the  following  : Neutral  ammonium 
chromate,  potassic  phosphate  and  sodic  sulphate  (Landois’  fluid, 
p.  26),  and  then  stain  it  in  bulk  for  24-48  hours  in  equal  parts  of 
the  above  solution  and  strong  arnmoniacal  carmine. 


xviil]  nerve-ganglia,  nerve-cells,  etc. 


219 


Squeeze  a little  of  the  red  pulp  between  two  cover-glasses,  and 
treat  it  as  recommended  for  the  methylene-blue  preparation.  It 
would  be  difficult  to  get  preparations  that  surpass  in  beauty  those 
prepared  by  the  methylene-blue  method  of  Thanhoffer. 

(iii.)  Make  a cover-glass  preparation  from  a fresh  spinal  cord. 
Heat  one  of  them  by  passing  it  two  or  three  times  through  the  flame 
of  a Bunsen-burner.  Thereby  the  proteids  are  coagulated  and 
partially  charred.  In  some  of  these  preparations  good  views  of  the 
blood-vessels  may  also  be  obtained. 

(a.)  Observe — especially  in  the  methylene-blue  preparation — the 
large  cells  with  numerous  branched  processes  ; some  of  them  are 
very  long,  and  each  shows  distinct  fibrillation. 

(/?.)  The  unbranched  axis-cylinder  process. 

(e.)  The  body  of  the  cell,  nucleated  and  nucleolated,  with  its  cell- 
contents,  traversed  by  blue-stained  fibrils,  running  in  certain  definite 
directions  through  the  cell. 

Other  forms  of  nerve-cells  are  referred  to  under  Cerebrum  and 
Cerebellum. 


ADDITIONAL  EXERCISES. 

11.  Motor  Nerves  to  Muscles. — (i.)  If  the  skin  over  the  sternum  of  a small 
frog  be  divided  longitudinally,  on  raising  the  skin  a small  thin  muscle — 
musculus  cutaneus  pectoris — will  be  seen  running  from  the  skin  to  the  sternum. 
Keep  the  muscle  stretched  and  “ fix  ” it  by  pouring  on  it  a little  osmic  acid. 
Cut  out  the  muscle,  and  after  dehydrating,  mount  it  in  balsam.  It  is  apt  to 
darken  on  exposure  to  light. 

(L)  Observe  the  nerve  is  black — sending  branches  over  the  muscular  fibres  ; 
trace  these  onwards  over  the  muscular  fibres  until  a single  nerve-fibre  is  found. 

(H)  Note  that  when  a nerve-fibre  divides,  it  does  so  always  at  a node  of 
Ranvier.  The  nerve-fibre  can  be  traced  to  a muscular  fibre,  but  it  apparently 
stops  abruptly,  because  the  myelin  stops  where  the  nerve  pierces  the  sarco- 
lemma.  Other  methods  are  required  to  see  the  termination  within  the  sarco- 
lemrna. 

(ii.)  May’s  Method. — Select  a thin  muscle,  e.gr.,  the  cutaneus  pectoris, 
sartorius,  mylo-hyoid,  &c.,  and  place  it  in  water  containing  2 per  cent,  glacial 
acetic  acid  for  twelve  hours.  Make — fresh — the  following  mixture  : — 

J per  cent,  potassio-gold  chloride  . . . i cc. 

2 ,,  osmic  acid  . . . . . i ,, 

2 ,,  glacial  acetic  acid  . . . • 50  ,, 

and  place  the  muscles  in  it  for  2-3  hours.  Then  transfer  them  to  the  following 
mixture  : — 

Glycerine 40  cc. 

Water  . . . . . . . . 20  ,, 

Hydrochloric  acid  (25  per  cent.)  . . . . i ,, 

for  several  hours.  They  become  very  transparent,  and  can  be  investigated  in 
glycerine  or  Farrant’s  solution. 


220 


PRACTICAL  HISTOLOGY. 


[XVIII. 


12.  Nerves  of  Frog’s  Sartorius. — Suj)pose  the  sartorius  to  be  selected.  A 
beautiful  view  of  the  distribution  of  the  motor  nerves — black — is  obtained 
(fig.  203). 

The  single  nerve-trunk  enters  the  muscle  on  the  median 
aspect  and  on  its  under  surface  about  the  level  between 
the  middle  and  lowest  thirds.  Several  large  branches — 
usually  two — run  nearly  parallel  towards  both  ends  of  the 
muscle — two  longer,  towards  the  upper  end  of  the  muscle 
A — and  two  or  three  shorter,  towards  its  lower  end,  the 
latter  following  a slightly  more  oblique  course.  Numerous 
branches  form  elongated  quadrilateral  meshes.  At  two 
points  in  the  muscle,  towards  its  ends,  there  are  more  fine 
branches  than  elsewhere.  The  fibres  form  plexuses  and 
divide.  Note  speciall}^  that  the  knee  and  pelvic  ends  are 
devoid  of  ncrve-fihres. 

13.  Motor  Nerve-Endings.— (i.)  Take  a thin  muscle, 
e.y. , the  eye-muscles  or  intercostal  muscles  of  a small 
mammal,  the  thin  leg  muscles  of  a lizard,  or  the  thin 
cutaneous  muscles  which  pass  between  the  skin  and  the 
wall  of  the  chest  in  snakes,  and  stain  them  with  gold 
chloride  by  the  formic  acid  gold  chloride  method  (p.  79). 
They  must  remain  in  the  gold  solution  about  one  hour. 
The  gold  may  be  reduced,  either  in  water  acidulated  with 
acetic  acid,  by  exposure  to  the  light,  or  in  the  dark,  in 
25  ]>er  cent,  formic  acid. 

(H)  Tease  a piece  of  the  purplish-violet  muscle  in 
glycerine,  and  search  for  a purple  nerve-fibre  termination 
in  an  arborescent  branched  end-plate  lying  on  the  sarcous 
substance  of  the  muscle  (fig.  204).  Nuclei  are  present  in 
the  protoplasm  of  the  end-plate. 

(ii.)  Golgi’s  Method. — Place  the  muscles  of  a newly- 
203.— Distribu-  killed  lizard  for  a minute  or  two  in  a . per  cent,  solution 


of  Nerve-  arsenic  acid,  and  directly  afterwards  in  a solution  of 


Fio. 
tion 

Fibres  in  the 
Frog’s  Sartorius. 
A.  Upper,  B. 

Lower  end ; aa 

and  bb.  Numer- 
ous fine  branches ; 
P.  Pelvic  end,  and 
K.  Knee  end,  with 
no  nerve-fibres. 


.5  per  cent,  solution  of  chloride  of  gold  and  potassium  for 
15-20  minutes,  and  reduce  the  tissue  in  sunlight  in  a i per 
cent,  solution  of  arsenic  acid.  Instead  of  the  above  gold 
solution,  use  the  following  mixture,  devised  by  Kiihne  : — 
Arsenic  acid  (.5  per  cent.)  . . . . 60  cc. 

Osmic  acid  (2  per  cent.)  . . . . 3 ,, 

Chloride  of  gold  and  potassium  (i  per  cent.) . 12  ,, 

The  tissue  is  then  placed  in  i per  cent,  arsenic  acid,  and  reduced  by  exposure 
to  sunlight.  The  process  may  be  greatly  hastened  by  doing  the  reduction 
process  at  a temperature  of  50°  C.,  but  it  must  be  done  in  the  direct  rays  of  the 
sun.  The  pieces  of  tissue  can  be  preserved  in  the  following  fluid,  devised  by 
Mays  : — 

Glycerine  . . . . ...  . 60  cc. 

Arsenic  acid  ( I per  cent. ) . . . . 10  ,, 

Methylic  alcohol 10  ,, 

Water  . . . . . . . 20  ,, 

In  working  with  solutions  of  gold,  do  not  use  steel  instruments.  They  must 
be  either  glass,  platinum-iridium,  or  the  substance  known  as  “ nickeline.” 

In  birds,  reptiles,  and  mammals  the  nerve-fibres  terminate  in  “ end-plates,” 
which  are  disc-shaped  bodies  lying  under  the  sarcolemma,  ^.c.,  they  are  h}*po- 
lommal  in  position,  40-60  fx.  long  and  40  ^ broad.  They  consist  of  a finely 
granular  protoplasm  with  nuclei.  As  the  gold  chloride  stains  only  the  axis- 


xviil]  nerve-ganglia,  nerve-cells,  etc. 


221 


cylinder,  one  sees  its  branched  arborescent  terminations  in  the  protoplasm 
(fig.  205).  A good  plan  to  see  the  unaltered  end-plates  is  to  examine  the 


Fig.  204.— Termination  of  a Nerve-Fibre  in  End-Plate  of  a Lizard’s  Muscle, 


muscle  in  a freshly-prepared  i j)er  cent,  solution  of  sulphate  of  iron  or  ammonio- 
sulphate  of  iron  {Kiiline,  Mays). 

14.  Frog’s  Motor  Nerve-Endings. — There  is  no  “end-plate,”  as  in  mamma- 
lian muscles,  but  the  axis-cylinder  splits  up  into  bayonet-shaped  branches 
under  the  sarcolemma.  The  myelin  is  continued  up 

nearly  to  the  sarcolemma,  but  as  the  ncrve-fibre 
perforates  the  sarcolemma  and  becomes  hypolemmal, 
the  myelin  stoj)s,  so  that  within  the  sarcolemma  the 
branches  are  pale,  and  consist  of  blanches  of  the  axial- 
C}dinder  only.  The  gold  method  (p.  79)  may  be  used. 

A simple  method  is  to  stain  a small  piece  of  a fre.sh 
muscle  {e.g.,  sartorius,  near  its  middle,  not  at  the  ends) 
with  Delafield’s  logwood.  It  stains  the  hypo-sarco- 
lemmal  nerve-terminations.  The  mode  of  termination 
of  nerves  in  sensory  surhices  will  be  found  under  Skin, 

Eye,  and  the  sense-organs  generally. 

15.  Terminations  of  Nerves  in  Tendon. — This  is 
best  shown  by  one  of  the  gold-chloride  methods. 

Perhaps  the  following  by  Manfredi  is  the  best : — 

Place  the  tendinous  ends  of  the  gemelli  muscles  of  a 
rabbit  (or  the  enucleated  eyeball  with  its  muscles 
attached)  in 


Potass  bichromate  (2  per  cent.)  . . for  3 days. 

Acetic  or  arsenic  acid  (i  per  cent.)  . ,,30  mins. 

Gold  chloride  ( I per  cent. ) . . . ,,30  ,, 


Fig.  205. — End-Plate  of 
a Lizard’s  Muscle. 
One  end  - plate  seen 
in  profile,  the  other 
from  above,  with  a 
nerve  - fibre  axis  - 
cylinder  terminating 
in  it.  Gold  chloride, 
Golgi’s  method. 


Wash  and  leave  exposed  in  sunlight  in  i per  cent,  arsenic  acid  until  it  assumes 
a violet-blue  colour. 

16.  Pyriform  Nerve-Cells  (Frog). — These  are  most  readily  found  in  the 
ganglion  of  the  vagus  as  it  issues  from  the  skull.  Pith  a frog,  distend  the 
oesophagus  by  pushing  a small  test-tube  into  it,  place  the  frog  on  its  belly, 
reflect  the  skin  over  the  shoulder-blade,  divide  the  trapezius  and  remove  the 
fore-limb.  The  vagus  will  be  seen  coming  out,  along  with  the  glosso-pharyn- 
geal,  through  a large  foramen  immediately  in  front  of  the  occipital  condyle. 
Clear  away  the  muscles  from  the  region  of  this  foramen,  snip  and  excise  a 


222 


PRACTICAL  HISTOLOGY. 


[XVIII. 


small  part  of  the  bone,  so  as  to  trace  the  nerve  as  far  back  as  possible.  Its 
greyish  gelatinous  semi-transparent  ganglion  is  seen.  Remove  the  nerve  with 
the  ganglion  and  place  it  for  twenty  minutes  in  i per  cent,  osmic  acid  and  then 
stain  it  in  picro-carmine.  Tease  a small  piece  in  glycerine,  and  it  is  by  no 
means  difficult  to  find  pyriform  cells,  each  with  a large  nucleus,  usually  near 
the  broad  end  of  the  cell.  The  protoplasm  frequently  contains  large  retractile 
granules.  The  straight  process  from  the  cells  is  readily  seen,  and  with  care 
the  spiral  process  also  can  be  seen. 

The  methylene-blue  method  (p.  192)  shows  very  well  the  straight  and  the 
spiral  fibre.  Place  the  fresh  tissue  in  the  methylene  fluid. 

17.  Isolated  Cells  of  Sympathetic  Ganglion  (Mammal). — (a.)  Tease  a sym- 
pathetic ganglion — best  done  after  maceration  for  24  hours  in  weak  acetic  acid 
(2  drops  to  100  cc.  water).  To  isolate  a cell  showing  its  connection  with  a 


nerve-fibre  requires  much  patience. 

{h.)  Place  a small  piece  of  the  superior 
cervical  ganglion  of  a rabbit  in  J per  cent, 
osmic  acid  for  2-3  hours.  Leave  it  in  water 
for  a day  or  two  to  macerate,  and  then 
tease  it  in  diluted  glycerine.  A better  plan 
is  to  make  an  interstitial  injection  of  the 
osmic  acid.  It  requires  to  be  carefully 
teased  to  get  isolated  cells  showing  several 
nerve-fibres  passing  off  from  them. 

(H)  Note  the  spherical  cell,  with  a 
nucleated  capsule.  It  gives  off  many  pro- 
cesses, each  of  which  becomes  continuous 
with  a nerve-fibre,  i.e.y  it  is  multipolar. 
These  cells  in  the  rabbit  usually  contain 
two  nuclei  (fig.  206). 

(c.)  Double  Impregnation  Method  of 
Ramon  y Cayal.  —It  is  better  to  use  the 
sympathetic  ganglia,  e.g.y  G.  stellatum  of 
embryos  (dog,  rabbit),  or  those  of  new-born 
animals.  The  fresh  ganglion  is  placed  in 
Golgi’s  bichromate-osmic  acid  fluid  (3  days), 
then  wash  it  with  distilled  water  and  after- 
wards with  .75  per  cent,  silver  nitrate. 
Leave  it  for  1-2  days  in  fresh  silver  nitrate 
(•75  cent.).  Wash  it  again,  and  place 
it  again  in  osmico-bichromate  mixture  (4- 
4J  days),  and  then  again  in  silver  nitrate. 
Sometimes  even  a “treble  im])regnatiou  ” is 
useful.  This  process — ^ ^ intensivo ''  or 
“ impregnacion  doble  ” — we  owe  to  R.  y 
Cayal,  who  has  obtained  good  results  with 
it,  and  so  has  L.  Sala,^  whose  paper ^ 
contains  figures  of  his  results  and  a resume 
of  the  literature.  He  finds  that  the  cells  are  multipolar  with  a single 
unbranched  proce.ss  and  numerous  branched  protoplasmic  processes. 

18.  Methylene -Blue  Method. — This  is  an  admirable  method,  and  depends 
on  the  fact  that  this  substance  stains  blue  the  axis-cylinders  or  fibrils  of 
nerves  m vivo.  It  is  applicable  for  studying  the  terminations  of  nerve-fibres 
in  any  tissue  where  they  terminate,  and  also  for  the  connection  between  nerve- 
fibres  and  nerve-cells.  When  injected  into  the  blood-vessels  of  an  animal,  it 


Fig.  206. — Isolated  Nerve-Cell  from 
Superior  Sympathetic  Ganglion  of 
a Rabbit,  ff.  Remak’s  fibres ; 
n'n'.  Nuclei  of  these  fibres ; nn. 
Nuclei  of  cell. 


^ Archiv  ital.  de  Biologic^  1893,  p.  439. 


THE  HEART  AND  BLOOD-VESSELS. 


223 


XIX.] 

acts  on  the  nerves,  and  on  the  latter  being  exposed  to  the  air  they  become 
blue.  It  may  also  be  applied  to  fresh  tissues. 

Inject  some  of  the  following  solution  into  the  blood-vessels  : — 

Methylene-blue  . . . . i gram. 

Normal  saline  ....  300  cc. 

Or  introduce  a 3 per  cent,  solution,  or  even  the  solid  substance,  into  the 
lymph-sac  of  a frog.  After  an  hour  or  two,  expose  a muscle  to  the  air, 
or  use  the  cornea,  or  any  other  tissue  with  nerves  in  it,  and  on  examining 
it  under  the  microscope  the  nerves  will  be  found  stained  blue.  To  preserve 
such  specimens,  mount  them  in  a solution  of  picrate  of  ammonia  and  glycerine 
(p.  192).  Fresh  tissues,  e.g.,  cornea  or  a thin  muscle,  may  be  immersed  in  a 
weak  solution  of  methylene-blue  with  the  same  result. ^ 

The  methylene-blue  metliod  may  be  used  for  the  study  of  nerve  terminations 
in  any  organ,  e.g.^  the  sense  organs,  and  in  arteries  one  can  see  most  beauti- 
fully the  plexus  of  non-medulhited  fibres  in  the  muscular  coat. 

19.  Nerve-Cells  of  Crayfish. — Select  a small  individual  and  inject  into  its 
abdominal  cavity  i to  2 cc.  of  a 0.2  percent,  solution  of  methylene-blue. 
After  8 or  10  hours,  remove  the  chitinous  covering  over  the  ganglia  ted  nerve- 
cord  and  expose  the  latter  in  a vessel,  which  admits  air  and  yet  prevents 
evaporation.  In  24  hours  or  so  excise  a ganglion  and  observe  it  in  a drop  of 
glycerine  tinged  with  picrate  of  ammonia  {Retzius). 


LESSON  XIX. 

THE  HEART  AND  BLOOD-VESSELS. 

Heart. — The  wall  of  the  heart  consists  of — (i.)  Pericardium; 
(2.)  Myocardium;  (3.)  Endocardium. 

The  pericardium  covering  the  heart  is  a serous  membrane 
composed  of  fibrous  tissue,  with  numerous  elastic  fibres,  and  covered 
on  its  free  surface  by  serous  endothelium.  It  is  sometimes  called 
epicardium.  The  fibrous  tissue  is  continuous  with  that  which 
invests  the  bundles  of  muscles  of  the  myocardium  itself.  Under- 
neath the  epicardium  are  the  blood-vessels,  nerves  (ganglia),  and 
the  lymphatics. 

The  myocardium  is  composed  of  striated  muscular  fibres,  whose 
characters  have  been  described  already  (Lesson  XVI.  12).  The 
fibres  are  arranged  in  bundles  separated  from  each  other  by  a 
greater  or  less  amount  of  connective  tissue,  in  which  run  the  blood- 
vessels and  nerves. 

The  endocardium  in  structure  resembles  the  pericardium,  but 
it  is  thinner.  It  consists  of  a fibrous  basis,  with  elastic  fibres 
covered  by  a single  layer  of  endothelium.  It  contains  a few  smooth 
muscular  fibres. 

An  artery  consists  of  three  coats  : — 

(i.)  Tumca  intima,  or  inner  coat,  composed  of  a single  layer 

^ S.  Mayer,  Zeitsch.  f,  wiss,  Mikros.,  vi.  422,  1889,  gives  numerous  references. 


224 


PRACTICAL  HISTOLOGY. 


[xix. 

of  endothelium  resting  on  an  elastic  lamina  composed  of  elastic 
networks,  or  an  elastic  membrane.  In  many  arteries,  however, 
there  is  a layer  of  connective  tissue  between  the  epithelium  and  the 
elastic  lamina — the  suh-epithelial  layer. 

(2.)  Tunica  media,  or  middle  coat,  consists  of  a varying  number 
of  layers  of  circularly-disposed,  short,  smooth,  muscular  fibres ; 
but  in  most  arteries,  and  chiefly  in  the  large  ones,  it  is  intermixed 
with  elastic  fibres  or  laminae. 

(3.)  Tunica  adventitia,  composed  of  fibrous  tissue  with  elastic 
fibres,  the  latter  especially  numerous  near  the  middle  coat. 

There  are,  however,  great  variations  in  structure  in  arteries, 
according  to  their  size  and  other  conditions. 

Veins. — Speaking  broadly,  the  veins  have  the  same  general 
structure  as  the  arteries.  They  are,  however,  much  thinner,  and 
some  of  them  have  valves.  They  consist  of  three  coats  ; the  inner 
coat  is  thinner  than  in  arteries,  and  the  elastic  lamina  thinner 


heart-fibres  ; c.  Cell ; n.  Nucleus  ; a.  Connective  tissue  ; «.  Vein. 

and  often  incomplete.  The  shape  of  the  endothelial  cells  is  different 
(fig.  213,  Y).  The  middle  coat  is  also  thinner,  and  has  less 
muscular  and  elastic  tissue,  and  relatively  more  connective  tissue. 
The  outer  coat  is  relatively  very  strong,  and  is  composed  of  fibrous 
tissue,  which  sends  processes  into  the  middle  coat.  There  are, 
however,  great  variations  in  the  structure  of  veins. 

Capillaries. — They  form  networks  of  fine  lubes  of  uniform 
diameter,  sufficient  to  allow  blood-corpuscles  to  pass  along  them 
freely  in  single  file.  The  arrangement  of  the  network  varies  in 
different  tissues.  The  walls,  when  examined  fresh,  appear  to  be 
homogeneous,  but  they  are  composed  of  flattened  epithelial  cells 
or  endothelial  cells  united  to  each  other  by  their  edges  by  cement 
substance,  which  is  blackened  by  silver  nitrate. 

1.  Heart. — Harden  small  pieces  of  the  heart  (human)  in  alcohol. 
Stain  a small  piece  in  bulk  in  borax-carmine,  and  then  place  it  for 
twenty-four  hours  in  acid  alcohol.  It  is  best  to  cut  sections  by  the 
paraffin  method  and  mount  them  in  balsam.  A very  instructive 


XIX.]  THE  HEART  AND  BLOOD-VESSELS.  22 S 

preparation  is  to  make  a transverse  section  across  both  ventricles  of 
the  heart  of  a small  mammal,  guinea-pig.  This  is  best  done 
in  a heart  stained  in  bulk. 

Sections  may  be  cut  by  means  of  a freezing  microtome,  and  then 
stained  with  picro-carmine,  and  mounted  either  in  Farrant^s  solution 
or  balsam.  In  both  cases  it  is  well  to  include  a section  of  the  peri- 
cardium and  also  of  the  endocardium.  Transverse  sections  of  the 
papillary  muscles  are  very  instructive. 

(a.)  (L)  Observe  the  branched  and  anastomosing  fibres,  but  in 
addition  some  of  them  will  be  cut  obliquely,  and  others  trans- 
versely, with  the  nuclei  stained  (comp.  p.  199). 

(b.)  (H)  The  faint  transverse  striation,  short  branches,  absence 
of  sarcolemma,  the  nucleus  in  the  substance  of  the  fibre,  and  the 
indistinct  cement  substance  (fig.  207). 

(c.)  The  fibrous  character  of  the  pericardium,  which  sends  fine 
septa  between  the  bundles  of  muscular  fibres.  If  the  pericardium 
be  not  included  in  the  section,  still  connective  tissue  will  be  seen 
between  the  bundles  of  fibres,  especially  in  transverse  sections  of 
these  (fig.  207,  B). 

2.  Purkinje’s  Fibres  (H)  occur  under  the  endocardium  in* the 
heart  of  the  sheep  and  some  other  animals.  Open  a ventricle  of  a 
sheep’s  heart,  observe  the  network 
of  fine  glistening  lines  j strip  off  the 
endocardium,  snip  out  a little  piece 
of  the  heart-muscle,  and  place  it  for 
thirty-six  hours  in  dilute  alcohol  or 
5 per  cent,  ammonium  chromate  for 
two  days.  Tease  a very  small  piece 
in  picro-carmine,  and  mount  in 
glycerine. 

(a,)  Search  for  isolated  polygonal 
cells,  each  with  usually  two  nuclei, 
and  the  edges  only  of  the  cells 
striated.  These  are  heart-cells  ap- 
parently arrested  in  the  process  of 
striation  (fig.  208). 

3.  Endocardium  (H). — Harden  a part  of  the  ventricle  of  the 
human  heart  in  alcohol  or  potassic  bichromate.  Make  sections  tc 
include  the  endocardium. 

{a.)  Observe  oval  nuclei  on  the  surface,  the  nuclei  of  the  endo- 
thelial cells.  Under  this  a superficial  layer  of  fibrous  tissue  (fig. 
209,  a),  with  a few  smooth  muscle-cells  (ml),  and  underneath  this 
fibrous  tissue  the  basis  of  the  membrane  (tc), 

(h,)  Outside  this  is  the  myocardium  (me). 

4.  T.S.  Heart-Valve  (H). — Harden  a cusp  of  a human  tricuspid 

21  o 


Fia.  20S. — Purkinje's  Fibres,  Dilute 
alcohol,  X 300. 


226 


PRACTICAL  HISTOLOGY. 


[xix. 


valve  in  chromic  and  spirit  fluid  (two  weeks),  make  transverse 
sections,  stain  in  logwood,  and  mount  in  balsam.  Alcohol  does 

well  as  a hardening  re- 
agent, and  the  sections 
can  then  he  readily- 
stained  in  picro-carmine. 

(a.)  On  the  surface, 
toward  the  auricle  (A), 
note  a superficial  layer 
of  lamellated  connective 

Fig.  2oq» — EndocRrdiimi  of  liCft  Vontnclc  ■fiaanp  ajvViipVi  iq 

a.  Superficial  layer;  ml.  Smooth  muscular  fibres;  Wmcn  IS  COVerea 

tc.  Fasciculated  fibrous  tissue;  me.  Muscle  of  with  endothelium  (fig. 
heart,  X 150.  2 10,  a,)  and  underneath 


this  a fibrous  basis  with  elastic  fibres  (re). 

(ft.)  If  the  section  passes  through  the  insertion  of  one  of  the 
chordae  tendineae,  it  presents  the  appearance  shown  in  fig.  210,  ct, 

5.  Aorta. — (i.)  Make  transverse  (and  longitudinal)  sections  of 
the  human  aorta,  or  of  that  of  an  ox,  which  has  been  hardened  in 


alcohol,  or,  preferably,  in  2 per  cent,  potassic  bichromate  (ten  days). 
Stain  a section  in  picro-carmine,  and  mount  it  in  glycerine. 

(ii.)  Another  good  method  is  to  slit  up  any  large  artery,  pin 


Fig.  210. — T.So  of  the  Cusp  of  the  Human  Tricuspid  Valve,  vertical  to  the  axis  of  the 
cusp.  A,  Auricular,  B,  Ventricular  surface;  a.  Superficial  lamellated  layer  of  the 
auricular,  and  a',  of  the  ventricular  surface ; ct  T S.  of  one  of  the  chordae  tendinese, 
where  it  is  inserted  into  the  valve  ; re.  Fibrous  tissue,  the  basis  of  the  valve,  x 100. 

it,  inner  surface  upwards,  upon  wood.  The  pins  must  be  close 
together  to  prevent  too  great  shrinking  of  the  tissue.  Place  it  in 
a dry,  well-aired  place,  say  near  a fire,  so  that  it  dries  within  a few 
hours.  Make  transverse  sections  with  a sharp  razor.  This  is  best 
done  by  making  a slit  in  a cork  and  clamping  the  dried  membrane 
in  the  slit.  Idace  the  sections  in  water;  they  swell  up  greatly. 
Remove  them,  stain  with  picro-carmine,  and  mount  in  formic 
glycerine. 


XIX.] 


THE  HEART  AND  BLOOD-VESSELS. 


227 


Fig.  21 1.  — L.S.  Human 
Thoracic  Aorta.  A.  In- 
ternal, B.  Middle,  and 
C.  External  coat,  x 20. 
Drying,  picro-carniine, 
and  acid  glycerine. 


(a.)  (L)  Observe  the  subdivision  into  three  coats  (fig.  211),  the 
tunica  intima  (inner),  media  (middle),  and  adventitia  (outer). 

(h.)  (H)  The  inner  coat  is  lined  by  a layer  of  squames,  -whose 
nuclei  may  be  detected  as  slight  oval  swellings  (not  seen  in  the 
dried  specimen).  Under  this  several  layers 
of  yellow  elastic  membrane,  with  a small 
amount  of  pink-stained  connective  tissue 
between  them.  The  outermost  layer  of 
elastic  membrane  is  generally  thicker  than 
the  others,  and  marks  the  outer  limit  of  this 
coat. 

(c.)  The  middle  coat^  composed  also  of 
numerous  elastic  laminae,  stained  in  this  case 
bright  yellow,  and  between  them  patches  of 
smooth  muscle  with  a somewhat  brownish 
tint,  and  some  connective  tissue  stained 
pink.  The  colour  of  these  two  tissues  is  quite  distinct. 

{d,)  The  outer  coat,  composed  of  white"  fibrous  tissue  (pink), 
some  elastic  laminae,  and  a few  smooth  muscular  fibres. 

It  is  obvious,  therefore,  that  elastic  tissue  enters  largely  into  the 
structure  of  the  larger  arteries. 

6.  Fenestrated  Membrane  of  Henle. — Tear  off  a thin  lamella 
from  the  inner  surface  of  a large  artery,  e.y.,  the  aorta  of  a sheep. 
Irrigate  it  with  acetic  acid,  or  place  it 
in  35  per  cent,  caustic  potash,  and 
mount  it  in  Tarrant’s  solution. 

(H)  Observe  the  elastic  laminae, 
some  of  them  with  holes  in  them 
(fig.  123).  These  laminae  tend  t8 
curl  up  at  their  edges.  (See  also 
Lesson  X.  7.) 

7.  T.S.  Medium- Sized  Artery,  e.g., 

the  femoral  artery  of  a child,  prepared 
and  stained  as  the  aorta. 

(a.)  (L)  Xote  the  three  coats. 

(6.)  (H)  The  inner  coat,  with  its 
endothelium  and  internal  elastic 
lamina.  In  many  arteries  a layer  of 
sub-epithelial  connective  tissue  lies  between  the  endothelium  and  the 
elastic  lamina.  The  elastic  lamina  is  thrown  into  folds  in  an  empty 
artery  owing  to  the  contraction  of  the  middle  coat  (fig.  212), 

{('.)  The  middle  coat  is  composed  of  several  layers  of  smooth 
muscle  arranged  circularly.  In  a Canada  balsam  preparation  the 
nuclei  stand  out  distinctly.  Amongst  the  muscle-fibres  are  twisted 
elastic  fibres  (fig.  212). 


M. 


Fig.  212. — T.S.  Artery.  I.  Tunica 
intima  ; M.  Media ; E,  Externa. 


228 


PRACTICAL  HISTOLOGY. 


[xix. 

{d.)  The  outer  coat,  chiefly  composed  of  white  fibrous  tissue 
intermingled  with  elastic  fibres,  some  of  which  are  cut  trans- 
versely. The  elastic  fibres  are  more  numerous  towards  the  inner 
part  of  this  coat.  Here  and  there  a section  of  a blood-vessel  may 
be  seen. 

8.  Endothelial  Lining  of  Veins  and  Arteries. — Cut  open  the 
external  jugular  vein  of  a rabbit  just  killed.  Pin  it  to  a piece  of 
cork — inner  surface  uppermost — by  means  of  hedgehog-spines. 
Wash  the  internal  surface  with  distilled  water,  and  then  a])ply 
to  it  for  five  minutes  ^ per  cent,  solution  of  silver  nitrate.  Wash 
off  the  silver,  place  the  vein  in  water  or  alcohol  and  water,  and 
expose  it  to  light.  Do  the  same  with  any  large  artery. 

(A.)  Snip  out  a small  portion  of  the  vein,  and  mount  it  in 
balsam,  inner  surface  uppermost. 

(H)  Observe  the  “silver  lines,”  indicating  the  existence  of  a 
layer  of  polygonal  squames  composing  part  of  the  inner  coat.  The 


Fia.  213.— A.  Epithelial  lining  of  an  artery  of  a calf,  and  V.  of  the  jugular  vein  of  a 
rabbit.  The  arrows  show  the  erection  of  the  blood-stream.  Silver  nitrate. 


long  axis  of  the  squames  lies  across  the  long  axis  of  the  vein  itself 
(fig.  213,  V). 

By  focussing  through  the  thickness  of  the  wall,  narrow  fusiform 
areas,  bounded  by  black  lines,  may  be  seen,  indicating  the  existence 
of  the  smooth  muscular  fibres  in  the  middle  coat. 

(B.)  W^ith  a razor  shave  ofl*  a thin  layer  from  the  brown  inner 
coat  of  the  artery.  Mount  it  in  balsam. 

(H)  Observe  the  elongated  lancet-shaped  endothelial  cells  (fig. 
213,  A);  the  long  axis  of  each  cell  in  the  long  axis  of  the  tube. 
The  variation  in  the  shape  of  the  epithelial  lining  of  vessels  seems 
to  have  relation  to  the  velocity  ‘of  the  blood-stream  in  these 
vessels. 

9.  Pia  Mater,  Capillaries,  Small  Arteries,  and  Veins  (H). — 

Carefully  remove  a small  piece  of  the  pia  mater  from  the  brain  of 
a sheep  recently  killed.  Lay  it  on  a glass  plate,  outer  surface 


THE  HEART  AND  BLOOD-VESSELS. 


229 


XIX.] 


lowest.  With  a camers-hair  pencil  dipped  in  normal  saline  solution 
brush  away  all  the  brain  matter,  leaving  a somewhat  villous-looking 
surface.  Cut  out  a small  part  of  the  membrane,  and  mount  it  in 
normal  saline. 

(a. ) Find  a capillary,  note  its  diameter,  uniform  calibre,  and  oval 
nuclei  bulging  slightly  into  the  lumen  of  the  tube. 

The  rest  of  the  wall  is  homogeneous  (%.  214). 

Trace  it  backwards  until  the  small  artery  or  arteriole 
with  which  it  is  continuous  is  found. 

{h.)  A small  artery  or  arteriole  with  a thin 
outer  coat,  and  a middle  coat  composed  of  a single 

layer  of  smooth  muscular  fibres  arranged  circularly  I'ig.  214.  — Capil- 
/ri  lanes  of  Brain, 

(hg,  216;.  ^ fresh,  X 100. 

(Co)  Select  a small  vein  which  is  somewhat 
thinner  than  the  corresponding  artery ; the  muscular  coat  is  very 
imperfect.  Irrigate  with  2 per  cent,  acetic  acid»  Nuclei  wherever 
present  come  distinctly  into  view. 

{d.)  The  oval  nuclei  of  the  lining  endothelium,  the  long  axis  of 


FiGo  215. — A.  A small  artery  with  the 
lumen  in  focus.  B.  Small  arteriole 
just  before  it  passes  into  a capil- 
lary, X 300. 


Fig.  216. — Small  Artery  from 
Human  Brain,  Pigment 
Granules  in  the  Adven- 
titia, X 150. 


the  nuclei  in  the  long  axis  of  the  vessel.  Outside  this  the  elastic 
lamina  appears . as  a somewhat  refractive  membrane  with  longi- 
tudinal folds. 

(e.)  The  vessel  crossed  transversely  by  the  long,  oval  nuclei  of 
the  muscular  fibres  of  the  middle  coat  (fig.  216),  Note  in  some 
cases  they  are  not  distributed  at  equal  distances,  but  in  groups 
(fig.  215).  One  point  must  be  particularly  studied,  viz.,  to  focus 
through  the  thickness  of  a small  vessel,  and  observe  carefully  that 
the  appearance  of  the  vessel  varies  with  the  position  of  the  lens, 
Le.,  whether  the  upper  surface,  lumen,  or  deeper  part  of  the  artery 
is  in  focus. 


230  PRACTICAL  HISTOLOGY.  [XIX. 

(/.)  In  the  outer  coat  the  elongated  fusiform  nuclei  of  the  con- 
nective tissue  cells  are  arranged  longitudinally. 

For  a permanent  preparation,  the  pia  mater  after  being  removed 
is  hardened  in  2 per  cent,  potassic  bichromate  and  preserved  in 
alcohol.  A thin  piece  is  selected  and  stained  with  logwood  or  with 
eosin-logwood,  and  mounted  in  balsam. 

10.  T.S.  Small  Artery  and  Vein.  — Select  a small  artery  and 
vein,  harden  in  MiilleFs  fluid,  stain  in  bulk  with  haematein,  and 
cut  in  paraffin. 

(H)  Observe  the  three  coats  in  each,  hut  they  are  much  thinner 
in  the  vein  than  the  artery.  In  the  former  the  intima  is  very  thin 
and  the  outer  coat  relatively  thicker  (fig.  217). 


Fig.  217. — T.S.  Small  Artery  and  Vein.  A,  Artery;  F.  Vein;  N.  Nerve. 


11.  Injection  of  Silver  Nitrate  into  Blood-Vessels. — (i.)  From 
the  aorta  inject  the  blood-vessels  of  a rabbit  with  .25  per  cent, 
silver  nitrate.  Before  doing  so  wash  out  the  blood-vessels  with 
normal  saline  to  remove  all  the  blood,  and  then  with  distilled 
water.  Slit  up  the  intestine,  wash  out  its  contents,  and  expose  it 
to  light  in  alcohol  and  water.  Scrape  away  the  mucous  membrane, 
leaving  only  the  muscular  coats.  Dehydrate  a small  piece  and 
mount  it  in  balsam.  It  is  easy  to  find  large  and  small  vessels  as 
well  as  capillaries. 

(a.)  (H)  Select  a small  artery,  and  note  in  it  the  endothelial 
lining  (fig.  219,  E)  and  the  circular  muscular  fibres  mapped  out 
from  each  other  by  silver  lines  (m). 


THE  HEART  AND  BLOOD-VESSELS. 


231 


XIX.] 

Arterioles  and  Small  Arteries. — (a.)  (H)  Select  a small  artery 
or  arteriole.  IsTote  the  layers  already  described.  If  the  circular 
muscular  fibres  be  arranged  in  one  layer,  note  that  the  fibres  are 
arranged  in  alternate  groups  on  opposite  sides  of  the  vessel  (fig, 
215,  A). 

(b,)  Trace  an  arteriole  into  a capillary,  and  note  the  change  in 
structure,  especially  how  the  muscle  disappears  (fig.  215,  B). 

(c.)  Select  a larger  vessel,  and  in  it  observe  the  structure  already 
described  and  shown  in  fig.  218. 

(d)  Find  capillaries,  and  note  the  endothelium  of  which  they 


Fig.  218.— Middle-sized  Ar- 
tery of  Brain,  a.  Endo- 
thelium; b.  Fenestrated 
membrane ; c.  Middle 
or  muscular  coat ; d. 
Adventitia ; e.  Pigment, 
X 300. 


Fig.  219. — Arteriole  of  the  Rabbit’s  Small 
Intestine.  E.  Endothelial  cells  of  the 
intima ; m,  Circular  muscular  fibres ; 
indicated  by  silver  lines,  x 200. 
Nitrate  of  silver. 


are  composed  (fig.  220),  In  order  to  see  the  nuclei  of  these  cells, 
the  preparation  should  be  stained  with  logwood. 

(ii.)  Instead  of  a rabbit,  the  blood-vessels  of  a frog  may  be 
injected  in  the  same  way  from  the  aorta  or  ventricle.  Use  a glass 
syringe.  Wash  out  the  vessels  first  with  normal  saline,  then  with 
distilled  water,  and,  finally,  inject  the  silver  nitrate  solution.  The 
bladder,  intestine,  and  mesentery  are  particularly  serviceable  for 
obtaining  small  vessels  and  capillaries. 

12.  Circulation  of  Blood. — A day  before  the  frog  is  required, 


232 


PRACTICAL  HISTOLOGYo 


[xix. 


let  the  brain  of  the  animal  be  destroyed.  An  hour  before  the  web 
is  to  be  examined,  place  two  or  three  drops  of  a . 5 per  cent,  watery 
solution  of  curare  in  the  lymph-sac  under  the  skin  of  the  animaPs 
back.  The  drug  should  not  act  too  rapidly.  After  a hour  or  so  it 
paralyses  the  extremities  of  the  motor  nerves, 
and  thus  makes  the  frog  motionless.  A small 
dose  of  the  drug  is  given  in  order  not  to 
affect  the  calibre  of  the  blood-vessels. 

Make  a frog-plate  by  taking  a piece  of 
stout  cardboard  or  thin  slip  of  wood  1 5 cm. 
long  (6  inches)  and  5 cm.  (2  inches)  broad. 
At  one  end  of  it  cut  a triangular  slit  whose 
base  is  2 cm.  or  less  in  width.  Tie  a thread 
round  the  tip  of,  e.^.,  the  third  and  fourth 
toes  of  the  hind-limb,  place  the  frog  on 
its  belly  on  the  board,  and  by  means  of 
the  two  threads  gently  stretch  the  web 
across  the  triangular  slit.  It  must  not  be 
^^ted  wiui^^iveS^  drawn  too  tightly.  The  threads  can  be  fixed 

in  slits  made  in  the  horns  bounding  the 
triangular  aperture.  To  prevent  evaporation  from  the  frog,  it  had 
better  be  placed  in  a moist  cotton  rag  or  surrounded  with  moist 
blotting-paper.  Moisten  the  web  with  a drop  of  water,  and  cover 
it  with  a narrow  fragment  of  a cover-glass.  In  selecting  a frog, 
choose  a light-coloured  one. 

(a.)  (L  and  H)  Find  an  artery,  and  note  that  the  blood  flows 
from  larger  into  smaller  vessels  with  what  appears  to  be  consider- 
able velocity.  Contrast  it  with  a vein,  in  which  the  blood  flows 
in  an  opposite  direction,  f.e.,  from  smaller  vessels — capillaries — to 
larger  ones,  but  the  current  is  slower  in  the  veins  than  in  the 
arteries.  The  walls  of  the  vein  are  slightly  thinner  than  those  of 
the  artery. 

(b.)  The  capillaries,  small  and  of  uniform  diameter,  with  the 
corpuscles  moving  in  single  file.  The  flow  is  uniform. 

(c.)  In  the  arteries  and  veins  note  the  rapid  red  central  stream, 
or  axial  zone  of  coloured  corpuscles,  and  next  the  wall,  on  either 
side,  the  peripheral  zone  or  space  of  Poiseuille,  narrow  and  free 
from  red  corpuscles,  but  containing  a few  white  ones  rolling  lazily 
along  the  vascular  wall  (H). 

(d,)  If  a red  corpuscle  happen  to  be  arrested  at  the  bifurcation 
of  a capillary,  other  corpuscles  impinge  on  it  and  bend  it.  As 
soon,  however,  as  it  is  dislodged  it  regains  its  shape,  so  that  the 
red  corpuscles  are  highly  elastic. 

(e.)  i^umerous  pigment-cells,  some  of  them  contracted,  others 
expanded,  are  visible  (fig.  144). 


THE  HEART  AND  BLOOD-VESSELS. 


233 


XIX.] 

If  desired,  the  phenomena  of  inflammation  can  readily  be 
studied  by  applying  some  irritant  to  the  web,  e.^.,  mustard  or 
creosote  (one  minute). 


ADDITIONAL  EXEECISES. 

13c  Elastic  Fibres  in  Arteries  {Martinotti). — Harden  the  blood-vessel  in 
chromic  acid,  make  sections,  and  stain  them  with  safranin  as  directed  in 
Lesson  X.  10.  All  the  elastic  fibres  are  purplish  or  black.  Herxheimer’s 
Method  (p.  161)  also  yields  good  results. 

14.  Development  of  Blood-Vessels. — (i.)  Harden  the  omentum  of  a newly- 
born  rabbit  in  Flemming’s  fluid  for  twenty-four  hours.  Wash  it  thoroughly  to 
remove  all  the  hardening  solution,  Stain  a piece  for  24-36  hours  in  safranin. 
Remove  the  surplus  stain  in  the  usual  way  with  acid  alcohol.  Mount  in 
balsam. 

(ii. ) Kill  a rabbit  five  days  old  with  chloroform  ; do  not  bleed  it.  Open 
the  abdomen,  remove  the  stomach  and  spleen,  and  attached  to  them  the 
omentum.  Place  all  in  a saturated 
watery  solution  of  picric  acid  for  one 
hour  ; wash  away  all  the  picric  acid,  cut 
out  a small  piece,  stain  it  in  logwood  and 
then  with  eosin,  or  double  stain  it  at 
once  in  eosin-hsematoxylin.  Mount  in 
FarranPs  solution. 

{a.)  (H)  Search  fora  network  of  capil- 
laries, which  is  easily  found.  Try  to 
find  one  of  them  which  gives  off  a long, 
narrow,  blunt  process.  The  process  may 
be  found  partially  channelled  (fig.  221). 

By  the  union  of  two  such  processes,  which 
ultimately  become  hollow,  new  capillary 
arches  are  formed.  The  blood-corpuscles  in  a preparation  of  (ii.)  are  stained 
with  eosin. 

15.  Nerves  and  Nerve-Cells  in  a Frog’s  Heart. — Pith  a frog,  expose  its 
heart,  cut  away  the  pericardium,  divide  the  frjenum  which  connects  the  pos- 
terior surface  of  the  ventricle  to  the  pericardium ; raise  the  heart,  find  the 
sinus  venosus,  ligature  the  inferior  and  two  superior  venae  cavae  which  open 
into  the  latter,  make  an  incision  into  one  of  the  aortae,  and  into  it  tie  a fine 
glass  cannula.  Inject  normal  saline  so  as  to  wash  out  the  cavities  of  the 
heart.  Distend  the  heart-cavities  with  the  following  mixture: — Four  parts 
of  gold  chloride  (2.  per  cent.)  and  one  of  formic  acid  boiled  together  and 
allowed  to  cool.  Ligature  the  other  aorta,  so  as  to  get  the  heart-cavities  fully 
distended.  Place  a ligature  below  the  cannula,  cut  out  the  heart  and  place  it 
for  J-i  hour  in  5 cc.  of  the  gold  mixture.  Open  the  auricles,  wash  out  the 
heai  t in  water,  and  expose  it  to  light  in  distilled  water — 50  cc. — containing 
three  drops  of  acetic  acid.  Reduction  of  the  gold  takes  place  slowly  in  3-4 
days.  Cut  out  the  auricular  septum  and  examine  it  in  glycerine. 

Pyriform  nerve-cells,  each  with  a straight  and  a spiral  process,  will  be 
found  along  the  course  of  the  nerves  in  and  near  the  auricular  septum. 

The  nerve-fibres  in  the  auricular  septum  are  readily  found  by  using  instead 
of  the  gold  a .2  per  cent,  solution  of  osmic  acid. 

The  Methylene-blue  method  yields  excellent  results,  if  the  fresh  auricular 


Fig.  221.— Capillaries  and  Developing 
Blood-Vessels  from  the  Omentum  of 
a New-Born  Rabbit.  Flemming’s 
fluid  and  safranin. 


234  PRACTICAL  HISTOLOGY.  [XX. 

septum  be  placed  in  a weak  solution  of  tlie  blue,  and  then  mounted  in 
ammonium  picrate  glycerine  (p.  192). 

16.  Circulation  in  the  Tongue  of  Frog. — Destroy  the  brain  of  a frog,  and 
after  a time  curarise  it  and  fix  it  on  its  back  on  a frog-plate  of  cork  with  a hole 
cut  in  it  just  in  front  of  the  head  of  the  animal,  the  hole  corresponding  in 
size  to  that  of  the  tongue.  The  tongue  is  attached  in  front,  and  it  can  thus 
readily  be  pulled  out  of  the  mouth  so  as  to  display  its  blood-vessels  from  the 
under-surface.  Pin  out  the  tongue  over  the  hole  in  the  cork,  when  the  under- 
surface of  the  tongue  will  be  uppermost.  Fix  the  tongue  under  the  microscope 
and  examine  it  (L).  One  can  study  the  circulation  in  its  vessels,  and  also 
observe  nerve-fibres  in  their  normal  condition. 


LESSOlf  XX. 

THE  LYMPHATIC  SYSTEM— SPLEEN— TONSILS— 
THYMUS  GLAND. 

The  lymphatic  vessels  have  thin  translucent  walls,  and  in  all 
essential  respects  resemble  veins  in  structure.  There  are  three 
coats  in  the  larger  vessels.  The  muscular  fibres  are  abundant  in 
the  middle  coat,  and  the  epithelium  of  the  inner  coat  is  in  some 
situations  sinuous  in  outline.  Valves  are  numerous  in  some 
situations.  The  larger  vessels  spring  from  so-called  lymph  capil- 
laries, which  are  usually  wider  than  blood-vessels,  and  they  unite 
with  each  other  and  form  an  irregular  plexus.  They  often  present 
dilations  and  constrictions,  and  consist  of  a single  layer  of  endothe- 
lium with  sinuous  outlines.  They  are  without  valves,  and  open  into 
the  smallest  regular  lymphatic  vessels. 

Lymphatic  Glands. — These  vary  much  in  shape,  size,  and  colour, 
but  they  are  frequently  oval  or  kidney-shaped.  At  one  part  is  a 
depression — the  hilum — where  the  medullary  part  of  the  gland 
comes  to  the  surface,  and  where  the  blood-vessels  enter  the  gland 
and  the  efferent  lymphatic  vessel  leaves  it.  There  is  usually  only 
one  efferent  vessel  and  several  afferent  lymphatics  ; the  latter  per- 
forate the  capsule  and  enter  the  gland  on  its  convex  side.  On 
making  a section  of  a gland,  with  the  naked  eye  one  can  see  that  it 
is  divided  into  a cortical  and  a medullary  part.  The  gland  is 
invested  by  a fibrous  capsule,  which  in  some  animals  contains 
smooth  muscular  fibres.  It  consists  of  two  layers,  the  outer  of 
coarser  and  the  inner  of  finer  connective  tissue.  It  sends  somewhat 
flattened,  large,  usually  unbranched,  septa  or  trabeculae  into  the 
cortex,  thus  dividing  it  into  a series  of  compartments  or  alveoli 


XX.] 


LYMPHATIC  GLANDS. 


23s 


(fig.  222).  These  trabeculae  are  continued  into  the  medulla,  where 
they  branch,  become  finer,  and  anastomose  to  form  an  irregular  net- 
work. The  lymphoid  tissue  lies  in  the  meshes  of  this  trabecular 
framework,  but  everywhere  separated  from  it  by  a lymph  sinus  or 
l5nnph  channel. 

In  the  cortex  the  alveoli  are  arranged  in  a regular  manner,  and 
the  greater  part  of  each  alveolus  is  occupied  by  a mass  of  adenoid 
tissue  crowded  with  leucocytes ; but  this  follicular  substance  is 
everywhere  separated  from  the  capsule  and  trabeculae  by  a lymph 
sinus,  traversed  by  a network  of  fine  fibrils  with  flattened  cells 
lying  on  them  at  the  points  of  intersection.  The  network 
is  coarser  than  that  of  the  adenoid  tissue,  and  it  contains  a few 
leucocytes.  It,  as  well  as  the  trabeculae,  is  covered  by  a layer  of 
sinuous  endothelium.  The  medulla  is  also  occupied  by  adenoid 
tissue  crowded  with  leucocytes,  but  the  lymphoid  tissue  forms 


Fig.  222. — L.S.  Cervical  lymph  glands  of  Dog.  c.  Capsule  ; s.  Lymph  sinus  ; F.  Follicle  ; 
a.  Medullary  cord  ; b.  Lymph  paths  of  the  medulla ; V.  Section  of  a blood-vessel ; 
HF.  Fibrous  part  at  the  hilum,  x 10. 


branching  or  anastomosing  cords — medullary  cords — each  one  sur- 
rounded by  its  lymph  sinus.  The  medullary  cords  are  continuous 
with  the  follicular  substance  in  the  cortical  alveoli.  Xear  the  centre 
of  the  lymphoid  tissue  of  the  alveoli  are  clearer  areas,  the  lymph- 
knots  or  germ-centres.  In  them  mitosis  goes  on  rapidly,  but  it 
requires  a high  power  to  discern  the  stages  of  the  division  of  the 
nuclei.  The  lymph  sinuses  are  continuous  throughout  the  gland, 
and  they  are  the  channels  through  which  the  lymph  moves.  Some 
glands  are  pigmented. 

LYMPHATIC  GLANDS. 

1,  Lymphatic  Glands. — Harden  in  5 per  cent,  ammonium 
bichromate  or  alcohol  the  lymphatic  glands  of  the  mesentery  of  a 
cat  or  calf,  or  those  found  in  the  neck  or  under  the  lower  jaw  of  a 


236 


PRACTICAL  HISTOLOGY. 


[xx. 


cat.  For  the  germ-centres  harden  a small  gland  in  Flemming’s  fluid 
and  stain  with  safranin.  Make  sections  including  both  poles  of  the 
gland  and  the  hilum.  Stain  them  with  logwood  and  then  with 
eosin.  Mount  in  balsam. 

(«.)  (L)  Observe  the  capsule  (fig.  222,  c)  surrounding  the  gland, 
and  sending  at  fairly  regular  intervals  septa  or  trabeculae  into  the 
substance  of  the  gland.  The  trabeculae  and  capsule  are  stained  by 
the  eosin.  The  trabeculae  are  flattened  in  the  outer  part  or 
cortex,  and  divide  it  into  compartments — follicles  or  alveoli  (F). 
The  trabeculae  are  continued  into  the  central  part  or  medulla, 
where  they  form  a network  of  smaller,  branched,  more  rounded 
trabeculae. 

(60)  The  compartments  in  the  cortex  are  nearly  filled  by  leuco- 
cytes lying  in  a meshwork  of  adenoid  tissue  constituting  the 
follicles  (cortical  nodules)  of  the  cortex  (fig.  222,  F).  Between 
the  trabeculae  of  the  medulla  the  leucocytes  are  equally  abundant. 
Owing,  however,  to  the  arrangement  of  the  trabeculae,  they  form 
medullary  cords,  but  everywhere  the  adenoid  tissue  is  continuous 
throughout  the  gland. 

(c.)  The  lymph  channels  (s)  exist  between  the  capsule  and  the 
follicles,  and  between  the  trabeculae  and  the  lymphoid  tissue  of  the 
cortex  and  medulla  (figs.  222,  223).  They  form  an  anastomosing 
system  of  paths  or  channels  throughout  the  gland,  and  are  traversed 
by  a fine  network  of  coarse  adenoid  tissue  with  comparatively  few 
leucocytes  in  its  meshes  (fig.  223). 

(<i.)  (H)  The  capsule  and  trabeculae,  chiefly  composed  of  connec- 
tive tissue,  and  in  some  animals  (ox)  with  smooth  muscular  fibres. 
Continuous  with  its  under  surface  is  a delicate  network  of  adenoid 
tissue,  which  stretches  across  the  lymph  channel  to  the  follicle, 
where  it  becomes  continuous  with  the  adenoid  tissue  supporting 
the  leucocytes  (fig.  223).  Between  the  trabeculae  and  the  medul- 
lary cords  in  the  cortex  similar  lymph  paths  with  an  adenoid  net- 
work. 

(e.)  The  follicles  and  medullary  cords,  everywhere  crowded  with 
leucocytes. 

2.  Adenoid  Reticulum. — (i.)  As  this  is  largely  obscured  by  the 
presence  of  the  leucocytes,  these  must  be  got  rid  of.  This  is  readily 
done  by  making  sections  of  the  lymph  gland  of  an  ox,  hardened  for 
two  or  three  days  in  5 per  cent,  ammonium  bichromate,  and  then 
shaking  the  sections  in  a test-tube  containing  water,  or  the  leuco- 
cytes may  be  “ pencilled  ” out  by  a camel’s-hair  pencil. 

(ii.)  A better  method,  perhaps,  is  to  inject  by  the  puncture 
method  (p.  237)  a ^ per  cent,  solution  of  silver  nitrate  into  the 
lymph  gland  of  an  ox.  The  fluid  being  driven  in  forcibly,  causes 
an  artificial  oedema  and  forces  the  parts  asunder.  Harden  in 


LYMPHATIC  GLANDS. 


237 


XX.] 


Fig.  223. — Lymph  Sinuses  from  the  Medulla  of  a 
Lymphatic  Gland.  T and  T.  Trabeculae  ; L. 
Lymph-path;  M.C.  Medullary  cord.  Silver 
nitrate  and  hsematoxylin,  x 300. 


alcohol,  stain  in  logwood  and  eosin,  and  mount  in  balsam.  The 
reticulum  in  the  ox  has  a brownish  appearance  from  the  deposition 
of  a brownish  pigment.  Sinu- 
ous outlines  of  endothelial 
cells  may  be  seen  on  the  tra- 
beculae, and  branched  cells  of 
the  lymph  sinus  (fig.  223), 
and  even  the  follicular  sub- 
stance. It  is  continuous  with 
the  endothelial  lining  of  the 
lympliatic  vessels. 

3.  Injection  of  the  Lymph 
Channels. — Fill  a hypoder- 
mic syringe  with  a watery 
solution  of  Berlin  blue,  force 
the  nozzle  of  the  syringe  into 
a small  lymph  gland  of  an 
ox,  and  inject  the  blue  fluid 
haphazard  into  the  gland.  The  blue  passes  into  the  lymph 
channels.  Harden  in  alcohol.  Make  sections  by  freezing,  stain 
them  with  picro-carmine,  and  mount  in  Farrant^s  solution. 

(L)  The  channels  are  filled  with  a blue  mass,  while  the  leuco- 
cytes are  red  and  the  septa  yellowish-red.  The  blue  mass  lies  under 
the  capsule,  and  in  the  lymph  paths  around  the  trabecula.  ISTotice 
the  difference  in  the  distribution  of  the  blue  mass  in  the  cortical 
and  medullary  parts  of  the  gland.  If  it  be  desired  to  study  the 
endothelium  covering  the  trabeculae,  inject  the  gland  as  above,  but 
with  per  cent,  silver  nitrate,  and  harden  in  alcohol. 

4.  Central  Tendon  of  Diaphragm. — Lave  the  central  tendon  of 
the  diaphragm  of  a newly-killed  rabbit  in  distilled  water.  Place  it 
for  an  hour  in  .2  per  cent,  silver  nitrate  in  a dark  place.  Remove 
it,  wash  again,  and  place  it  for  twenty-four  hours  in  water  contain- 
ing a little  alcoholic  solution  of  thymol  (10  per  cent.),  or  a drop 
of  carbolic  acid  to  prevent  the  formation  of  fungi.  Maceration  in 
water  enables  the  endothelium  on  the  surface  to  be  readily  pencilled 
off.  Mount  it  in  balsam. 

(L)  Examine  the  pleural  surface  with  the  naked  eye  or  with  a 
lens,  and  a plexus  of  lymphatic  vessels — clear  on  a dark-brown 
ground — will  be  seen  (fig.  224).  The  vessels  anastomose,  and  lead 
into  narrow  vessels,  which  run  more  or  less  parallel  to  each  other, 
and  correspond  to  inter-tendinous  spaces. 

(H)  Observe  the  dilations  and  constrictions  in  the  finer  lymphatic 
vessels,  and  their  sinuous  epithelium  (fig.  225,  L),  and  the  com- 
munications between  the  cell-spaces  and  the  lymphatics.  (Lesson 
XL  12.) 


238  PRACTICAL  HISTOLOGY.  [XX. 

u.  Septum  Cistemse  Magnae  Lymphaticse. — Open  the  abdomen 


Fig.  224. — Lymphatic  Vessels  of  the  Central  Tendon  of  the  Diaphragm  of  a Rabbit,  the 
lining  endothelium  shown.  Silver  nitrate. 

of  a newly-killed  frog  and  remove  the  intestines.  Turn  the  frog 


Fig.  225. — Pleural  Surface  of  the  Central  Tendon  of  Diaphragm  of  Rabbit.  L.  Lymphatic 
vessel  lined  with  sinuous  endothelium ; c.  Cell-spaces  of  connective  tissue.  Silver 
nitrate,  x no. 


on  its  belly.  Cut  through  the  vertebral  column  at  its  lower  end, 


LYMPHATIC  GLANDS. 


239 


XX.] 

raise  it,  and  cut  out  a square  window — about  | inch  in  diameter, 
including  the  whole  of  the  posterior  body-wall.  This  exposes  the 
dorsal  surface  of  a thin  membrane,  the  septum  of  the  great  lymph- 
sac.  It  is  attached  to  the  kidneys  on  each  side.  Pin  the 
membrane,  by  means  of  hedgehog-spines,  to  a thin  ring  of  cork 
with  a hole  in  it.  Wash  the  membrane  with  distilled  water  and 
place  it  for  ten  minutes  in  .5  per  cent,  silver  nitrate  solution.  Wash 
it — still  on  its  ring  of  cork — in  distilled  water,  and  expose  it  to 
light.  After  it  has  become  brown,  cut  it  into  pieces,  and  mount 
them  in  Farrant^s  solution,  one  with  the  peritoneal  surface  upper- 
most, the  other  with  its  dorsal  surface  uppermost. 

A,  (H)  Peritoneal  Surface. — Observe  the  slightly  sinuous 
“silver  lines,”  indicating  the  existence  of 

a single  layer  of  endothelium.  Here  and  , 

there  small  stomata  or  openings,  which 

lead  from  the  peritoneal  cavity  to  the  great 

lymph-sac  (fig.  226).  The  stomata  may 

be  closed  or  open,  and  are  recognised  by 

their  brownish  appearance.  They  are  sur- 

rounded  by  a few  finely-granular  brown-  ? \J 

stained  cells  — germinating  epithelium. 

The  pointed  angles  of  several  of  the  larger 
endothelial  cells  radiate  from  these  aper- 

Focus  through  the  thickness  of  the 
membrane,  and  note  its  fibrous  character.  Fig.  226.  — Endothelium  and 
On  the  deeper  surface  another  layer  of  |uXfe^ofthe®Septum 

endothelial  cells  comes  into  view.  They  tern®  Lymphaticse  Magnse 
. 1 j.T_  of  the  Frog,  x 200. 

are  more  sinuous  and  narrower  than  those 
on  the  peritoneal  surface. 

B.  The  Dorsal  or  Cisternal  Surface. — Observe  the  more  sinuous, 
polygonal,  and  broader  endothelial  cells  covering  this  surface  of 
the  membrane ; and  the  other  openings  of  the  stomata. 

The  serous  cavities  communicate  hy  means  of  stomata  with  the 
lymphatic  system.  Stomata  occur  on  the  pleura,  under-surface  of 
the  diaphragm,  and  mesentery. 


THE  TONSILS. 

6.  Tonsils. — Use  those  of  rabbit  or  cat,  as  the  human  tonsil 
does  not  give  such  good  results.  Fix  them  in  Kleinenberg’s 
fluid  (12  hours),  and  then  harden  in  gradually-increasing  strengths 
of  alcohol.  Or  use  mercuric  chloride  or  Flemming’s  fluid  (3-6 
hours).  Make  sections  by  the  paraffin  method,  and  stain  them 


240 


PRACTICAL  HISTOLOGY. 


with  logwood  or  logwood  and  eosin,  and  mount  in  balsam.  Sections 
may  be  stained  first  in  acid-haematoxylin  and  then  counter  stain 
them  with  eosin.  For  the  lymph-knots  or  germ-centres  stain  with 
safranin  and  counter  stain  with  picric  acid. 

For  fixing  the  sections  on  a slide,  Gulland  ^ uses  a modification  of 
the  capillary-attraction  method  of  Gaule.  The  sections  in  paraffin 
are  placed  in  warm  water  as  recommended  by  Gaskell^  Float  the 
sections  on  to  a slide,  pour  off  the  surplus  water,  and  expose  the 
slide  for  several  hours  to  a temperature  under  that  of  the  melting- 
point  of  the  embedding  paraffin.  When  dry  remove  the  paraffin 
by  xylol  and  then  stain  the  sections.  They  adhere  to  the  slide  by 
capillary  attractiom 

(L)  Observe  on  the  surface  the  stratified  squamous  epithelium, 
and  under  it  numerous  round  or  oval  aggregations  of  adenoid  tissue 
(fig.  227).  These  form  but  imperfect  nodules.  Pit-like  recesses 
are  seen,  lined  by  stratified  epithelium,  and  into  them  mucous  glands 
sometimes  open. 

(H)  Trace  some  of  the  leucocytes  of  the  adenoid  tissue  upwards 


Tig.  227. — Single  Follicle  of  Tonsil,  x 20.  i.  Cavity  of  follicle  ; 2.  Epithelium  infil- 
trated with  leucocytes;  3.  Adenoid  tissue ;/i,  2,  and  3.  Follicles  cut  in  various 
directions,  /i  with  a lymph-knot ; 4.  Fibrous  sheath ; 5.  Section  of  duct  of  mucous 
gland ; 6.  Blood-vessel. 

between  the  epithelial  cells,  so  that  the  epithelial  layer  is  at  places 
infiltrated  with  leucocytes. 


1 Jour,  of  Anat,  and  Phys,^  xxvi.,  1891,  p.  56. 

* Quart.  Jour,  Micros,  Sci,,  xxxi.,  1891,  p.  382, 


XX.] 


THE  THYMUS  GLAND. 


241 


THE  THYMUS  GLAND. 

This  gland  is  very  large  in  the  embryo  and  infant,  hut  it  begins 
a retrograde  development  about  the  sixth  year,  and  is  eventually 
replaced  by  fat  and  connective  tissue.  In  the  rabbit  it  retains  its 
structure.  It  is  composed  of  a number  of  lobes,  and  these  again  of 
smaller  lobules.  A capsule  composed  of  connective  tissue  holds 
all  together,  and  sends  in  septa — carrying  blood-vessels  and  lym- 
phatics— between  the  lobes  and  lobules,  and  also  fine  prolongations 
into  the  interior  of  the  latter.  There  are  no  smooth  muscular  fibres 
in  tlie  septa.  Each  lobule  consists  of  a cortical  and  a medullary 
part.  Within  each  lobule  is  a delicate  network  of  reticular  con- 
nective tissue,  finer  and  more  like  adenoid  tissue  in  the  medulla. 
It  appears  to  consist  of  branching  cells,  and  is  coarser  in  the  cortical 
part.  The  meshes  are  crowded 
with  leucocytes,  which,  however, 
are  most  abundant  in  the  cortex. 

The  medullary  substance  contains 
the  concentric  corpuscles,  like 
nests  of  concentrically-arranged 
flattened  epithelial  cells  (fig.  228). 

The  blood-vessels  run  along  the 
septa  and  form  a capillary  plexus 
within  the  lobules. 

7.  Thymus  Gland. — Harden 
the  thymus  of  a young  animal 
or  child  in  Muller’s  fluid  (three 
weeks),  and  then  in  gradually- 
increasing  strengths  of  alcohol  Sections  stained  with  logwood  are 
mounted  in  balsam. 

(a.)  (L)  Observe  the  capsule  sending  septa  between  the  larger 
lobules,  and  finer  septa  into  the  lobules,  thus  subdividing  them  into 
smaller  secondary,  lobules.  Each  such  small  lobule  is  about  1 mm. 
in  diameter,  and  as  one  is  exactly  like  the  others,  it  suffices  to 
study  one. 

(b.)  A darker,  denser  peripheral  zone,  the  cortex,  and  a more 
open  light  central  part  or  medulla,  the  former  surrounding  the  latter 
(fig-  228). 

(c.)  (H)  The  septa  consist  of  fibrous  tissue  with  some  elastic 
fibres,  with  numerous  blood-vessels  and  slits  ; the  latter  are  the 
lymphatics.  The  lobule  consists  of  adenoid  tissue — the  mesh- work 
not  visible  because  it  is  crowded  with  leucocytes. 

22 


228  — Section  of  a Few  Lobules  of  a 
Child’s  Thymus.  C.  Cortical,  M.  Medul- 
lary part;  c.  Concentric  corpuscles, 
X 20. 


Q 


242 


PRACTICAL  HISTOLOGY, 


[xx. 


{d.)  A variable  number  of  coiicentrically-striated  bodies,  con- 


centric corpuscles  or  HassalFs  corpuscles.  Sections  of  capillaries 
amongst  the  adenoid  tissue  (fig.  230). 


The  spleen,  like  the  thymus,  thyroid,  and  some  other  glands, 
is  a “ ductless  gland,’’  and  is  invested  by  a fibrous  capsule.  The 
capsule  consists  of  an  outer  layer  of  connective  tissue  covered  by 
endothelium — the  serous  or  peritoneal  covering — and  a deeper 
layer  of  connective  tissue  with  networks  of  elastic  fibres,  and  in 
some  animals  (dog,  cat,  pig)  smooth  muscular  fibres.  From  the 
deeper  surface  of  the  latter  flattened  or  rounded  trabeculae  pass  into 
the  organ,  and  as  they  do  so  they  branch  and  anastomose,  thus 
forming  a spongy  mesh  work  with  a labyrinth  of  communicating 
spaces.  These  spaces  are  filled  with  a reddish-purple  soft  substance, 
the  splenic  pulp.  The  blood-vessels  are  ensheathed  by  connective 
tissue,  to  which  the  trabeculae  are  attached.  The  red  colour  of  the 
pulp  is  due  to  the  large  number  of  blood-corpuscles.  The  trabe- 
cular framework  is  continuous  with  the  connective  tissue  entering 
the  organ  along  with  and  covering  the  blood-vessels — “ adventitial 
sheath  ” — at  the  hilum  of  the  organ.  In  the  splenic  pulp  are  small 
spherical  whitish  bodies — Malpighian  corpuscles  (0.2-0. 7 mm.). 
The  Malpighian  corpuscles  are  small  groups  of  leucocytes  de- 
veloped here  and  there  in  the  adventitia  of  the  splenic  artery. 


Fig.  229.— Injected  Lobules  of  Thymus 
of  a Cat.  a.  Cortex  ; h.  Medulla  ; 
c.  Blood-vessels ; d.  Septum  of 
connective  tissue. 


Fig.  230.— Elements  of  the 
Thymus  Gland,  a.  Leu- 
cocytes ; h.  Concentric 
corpuscle,  x 300. 


THE  SPLEEN. 


THE  SPLEEN. 


243 


XX.] 

In  the  guinea-pig  the  lymphoid  mass  lorms  almost  a continuous 
covering. 

The  Malpighian  corpuscles  occur  chiefly  at  the  bifurcations  of 
the  artery,  so  that  the  artery  perforates  them  usually  at  one  side, 
and  thus  the  mass  is  arranged  in  a lob-sided  manner  on  the  artery. 
In  structure  they  resemble  the  follicular  substance  of  lymphatic 
glands,  and  in  the  centre  of  some  of  them  is  a lymph-knot,  in  which 
mitosis  occurs. 

The  splenic  pulp  consists  of  a mesh- work  composed  of  branched 
cells  with  membranous  expan- 
sions, and  the  processes  of 
neighbouring  cells  anastomose 
to  form  a fine  reticulum,  which 
occupies  the  irregular  chambers 
of  the  trabecular  framework, 
and  with  which  it  is  continu- 
ous. The  meshes  of  this  fine 
network  are  occupied  by  cellu- 
lar elements  in  considerable 
variety.  This  reticulum  is  per- 
meated by  blood-corpuscles — iu 
fact,  the  blood  stream  of  the 
spleen  seems  to  bear  the  same 
relation  to  this  reticulum  that 
the  lymph  stream  bears  to  a 
lymph  gland.  Besides  red 
blood  - corpuscles  there  are 
larger  cells  called  splenic  cells ; 
some  of  these  often  contain 
degenerated  blood-corpuscles  or 
pigment.  There  are  also  leuco- 
cytes (fig.  232).  The  arteries 
enter  the  spleen  at  the  hilum, 
and  run  for  a short  part  ' of 
their  course  in  the  trabeculse, 
which  they  soon  leave,  enter  the  reticulum,  and  break  up  into 
pencils  or  groups  of  small  arteries.  Some  of  these  open  into  true 
capillaries  in  the  Malpighian  corpuscles ; other  fine  branches  open 
directly  into  the  reticulum.  The  endothelial  lining,  instead  of 
forming  a continuous  membrane,  leaves  apertures  between  the  cells, 
through  which  the  blood  escapes.  The  veins  arise  from  the  spaces 
of  the  reticulum,  and  rapidly  pass  into  the  trabeculae,  in  which  they 
are  firmly  fixed.  Near  the  hilum,  and  for  part  of  their  course,  the 
arteries  and  veins’lie  in  a ‘‘  common  sheath,’’  with  the  corresponding 
nerves.  The  vein  is  always  much  wider  than  the  corresponding 


244 


PRACTICAL  HISTOLOGY. 


[xx. 


artery.  The  lymphatics  are  not  very  numerous.  For  the  nerves 
see  Lesson  XYII.  7. 

8.  Spleen. — Tie  the  blood-vessels  at  the  hilum  of  the  spleen  of  a 
cat,  so  as  to  keep  the  blood  in  the  spleen.  Cut  it  out  and  place  it 
in  a large  volume  of  Muller’s  fluid  (two  weeks)  or  2 per  cent, 
bichromate  of  potash.  Wash  it  thoroughly  in  running  water  for  an 
hour  or  two.  Cut  out  small  pieces  and  harden  them  in  alcohol. 
Make  transverse  sections,  ^.6.,  across  the  long  axis  of  the  organ ; 
stain  one  in  logwood,  and  mount  in  balsam.  Other  sections  are  to 
be  stained  in  picro-carmine  and  mounted  in  Farrant’s  solution,  and 
a thin  set  placed  in  i per  cent,  osmic  acid  (twenty-four  hours),  and 
mounted  in  Farrant’s  solution.  This  sharpens  the  outlines  of  the 
elements.  Stain  other  sections  in  eosin-logwood  or  safranin  (forty- 
eight  hours). 

9.  T.S.  Spleen  (L)  {Cat;  liceynatoxylin  or  eosin  ^ndilmmatoxylin). 
— The  Malpighian  corpuscles  are  visible  as  small  blue  spots  to  the 
naked  eye. 

(a.)  Externally  the  capsule,  fibrous,  thick,  firmly  adherent  and 
closely  applied  to  the  organ,  sends  trabeculae  into  the  spleen,  where 
they  branch  and  anastomose  to  form  a trabecular  framework.  Some 
of  them  will  be  cut  longitudinally,  others  obliquely,  and  some  trans- 
versely. In  the  larger  trabeculae,  sections  of  large  blood-vessels 


(fig.  231).  Xote  that  there  is  no  lymph  space  between  the  capsule 
and  the  gland  substance,  as  is  the  case  in  lymph  glands. 

{h.)  Filling  the  interstices  of  this  network,  the  splenic  pulp,  and 
in  it  oval  or  rounded  bodies— Malpighian  or  splenic  corpuscles — 
as  blue-stained  bodies  contrasting  with  the  yellowish  brown  pulp  in 
which  they  lie.  In  the  centre  of  each  is  a lighter  area,  the  ‘‘germ- 
centre”  of  Flemming.  In  each  corpuscle  a section  of  a small 
artery  lying  excentrically  in  the  mass.  The  splenic  corpuscles  are 


Fig.  232.— Elements  of  Human  Splenic 
Pulp.  1.  Leucocytes;  2.  Epithelial 
cells;  3.  Coloured  blood-corpuscles; 
4.  Cells  containing  pigment-granules. 


Fig.  233. — Reticulum  of 
the  Splenic  Pulp. 


XX.] 


THE  SPLEEN. 


245 


small  lob-sided  aggregations  of  lymphoid  tissue  around  branches  of 
the  splenic  artery.  They  are  relatively  more  numerous  than  in  the 
human  spleen.  The  track  of  the  blood  in  the  pulp  is  mapped  out 
by  ^he  yellow  blood-corpuscles. 

(c.)  (H)  The  capsule  and  trabeculae,  composed  of  fibrous  tissue, 
with  elastic  fibres  and  smooth  muscle.  The  Malpighian  corpuscles, 
consisting  of  leucocytes  in  an  adenoid  reticulum.  The  centre  is 
lighter  in  tint  than  the  circumference,  which  is  more  condensed. 
The  lighter  centre  is  due  to  the  larger  cells  present  there.  They 
are  undergoing  proliferation.  The  cells  formed  in  the  splenic 
corpuscles  pass  into  the  spaces  of  the  pulp  and  leave  the  organ  by 
the  venous  blood  stream. 

(d.)  In  the  pulp  irregular  rows  of  coloured  blood-corpuscles — 
yellow — and  between  these  leucocytes  and  other  cellsr 

The  exact  structure  of  the  pulp  can  only  be  properly  studied  in 
a section  which  is  very  thin,  and  especially  at  the  edges  of  the 
section,  or  best  of  all  in  a section  of  a dog’s  (or  cat’s)  spleen,  whose 
blood-vessels  have  been  washed  out,  and  cleared  of  all  blood- 
corpuscles  by  a warm  stream  of  normal  saline  solution.  The 
vessels  are  then  injected  with  a 5 per  cent,  solution  of  ammonium 
bichromate,  and  the  organ  hardened  in  a large  quantity  of  the  same 
fluid,  and  subsequently  in  alcohol. 

(e.)  In  a section  prepared  in  this  way,  or  at  the  edges  of  a very 
thin  section,  the  fine  reticulum  of  branching  cells  may  be  seen 
(fig.  233)  with  the  cells  of  the  splenic  pulp  washed  out  of  it. 

10.  Human  Spleen. — Harden  this  in  the  same  way  as  8.  !N"ote 
that,  as  a rule,  the  Malpighian  corpuscles  are  less  numerous.  In 
other  respects  the  ^general  structure  is  the  same. 

11.  Injected  Spleen. — It  is  very  difficult  to  inject  the  finer  splenic 
blood-vessels.  They  should  be  washed  out  first  with  normal  saline, 
and  preferably  a watery  solution  of  Berlin  blue,  or  Berlin  blue  with 
gelatine,  should  be  used  as  the  injection.  Note  that  an  artery  and 
capillaries  exist  in  the  Malpighian  corpuscles,  but  the  splenic  pulp 
seems  to  be  infiltrated  with  a blue  mass.  The  capillaries  open  into 
this  system  of  labyrinthine  blood-passages.  These  intermediate 
blood-passages  are  merely  the  spaces  amongst  the  cells  of  pulp  and 
are  not  lined  by  epithelium.  The  terminations  of  the  capillaries 
in  some  situations  are  surrounded  by  thick  sheaths  or  collars  of 
tissue,  perha[)S  derived  from  the  cells  of  the  pulp  {Banmeavth)} 

12.  The  varieties  of  Leucocytes  in  lymph  glands  are  best  studied 
by  fixing  the  gland  in  Hg  CI2  and  staining  sections  with  Ehrlich- 
Biondi  fluid  {hloyer)?  There  are  at  least  four  varieties,  not  including 
phagocytes. 

^ Archiv f.  mile.  Anat,^  xxxviii.  p.  345.  ^ Ibid.,  xxxiv. 


246 


PKACTICAL  HISTOLOGY. 


[XXI. 


LESSON  XXL 

TONGUE— TASTE-BUDS— SOFT  PALATE. 
TONGUE. 

Place  small  portions  in  Muller’s  fluid  or  2 per  cent,  potassic 
bichromate  for  fourteen  days,  and  complete  the  hardening  in 
alcohol,  or  harden  it  in  mercuric  chloride.  Make  vertical  trans- 
verse sections^  It  is  well  to  have  the  tongue  of  a small  cat  or 
kitten,  and  parts  of  the  human  tongue  also — the  former  because  a 
complete  transverse  section  can  be  put  on  a slide.  The  structure 
will  vary  according  as  the  section  is  made  through  the  anterior  or 
posterior  part  of  the  organ,  as  the  latter  contains  many  lymph 
follicles  and  mucous  glands.  The  sections  may  be  stained  in 
logwood  and  mounted  in  balsam. 

1.  T.S.  Tongue  of  Cat. — (a.)  (L)  Observe  the  papillae,  of  various 
shapes,  on  the  dorsum  of  the  tongue,  and  covered  by  stratified  epi- 
thelium. Under  this  the 
connective  tissue  of  the 
mucous  membrane  (fig. 

234)- 

(b,)  Muscular  Fibres. — 

Many  cut  transversely  and 
arranged  in  groups  under 
the  dorsal  mucous  mem- 
brane and  elsewhere ; others 
which  run  from  the  vertical 
mesial  plane  or  septum  hori- 
zontally outwards,  and  sonie 
which  pass  vertically.  Th^\^ 
last  may  be  seen  to  become 
conical  and  end  in  the  connective  tissue  of  the  mucous  membrane. 
Some  of  these  fibres  branch.  (The  methods  of  isolating  branched 
fibres  are  referred  to  in  Lesson  XYI.  3.) 

(r.)  Lingual  Papillae. — The  dorsum  of  the  tongue  is  beset  with 
elevations* of  the  mucous  membrane  covered  by  stratified  epithelium, 
and  constituting  three  varieties  of  papillae. 

(i.)  Filiform  (.7-3  mm  long),  by  far  the  most  numerous,  and  are 
placed  all  over  the  dorsum.  They  are  conical  elevations  of  the 
mucous  membrane,  the  upper  end  of  which  is  beset  with  five,  fifteen, 


Fig.  234  — T S.  ot  One-halt  of  the  Tongue  of  a Cat. 


XXI.] 


TONGUE. 


247 


or  thirty  secondary  papillae.  Each  papilla  is  composed  of  fibrous 
tissue  with  elastic  fibres,  and  covered  by  many  layers  of  stratified 
epithelium,  the  superficial  cells  of  which  are  often  corneous  (fig.  235). 

(2.)  Fungiform  (0.5-1. 5 mm.  long),  are  not  nearly  so  numerous 
as  the  foregoing,  and  are  scattered  over 
the  do  rsum.  Each  papilla  is  club-shaped 
or  lenticular,  with  a constricted  base. 

The  apex'  is  beset  with  secondary 
papillae,  but  the  epithelium  covering 
them  is  thinner  than  in  (i)  (fig.  236). 

(3.)  Circumvallate  (1-1.5  mm.  high 
and  1-3  mm.  broad),  are  confined  to 
the  posterior  part  of  the  tongue,  where 
they  (8-15  in  number)  are  arranged  in 
the  form  of  a V,  the  apex  of  the  V 
being  directed  backwards.  Each  circu- 
lar elevation  is  raised  above  the  level 
of  the  tongue  and  surrounded  by  a 
circular  trench  or  fossa.  Secondary 
papillae  occur  only  on  their  surface. 

Taste-bulbs  occur  in  the  wall  of  the 
papilla  directed  toward  the  fossa.  They 
are  the  organs  of  taste,  and  are  supplied 
by  the  glosso-pharyngeal  nerve.  It  may  require  several  sections  to 
obtain  views  of  all  three  forms  of  papillae. 

(d,)  (H)  The  stratified  squamous  epithelium  covering  the  papillae 
and  sides  of  the  tongue. 

The  superficial  cells  are 
very  thin. 

(e.)  Glands,  in  the 
back  part  mucous 
glands  with  clear  con- 
tents, and  it  may  be 
also  serous  glands  in 
which  the  acini  are 
more  granular  (fig 

237)- 

(/)  Fat-cells,  like 
padding  between  the 
striped  muscular  fibres 
here  and  there.  Hear 
the  lower  surface,  sections  of  the  lingual  artery  and  nerves.  In  the 
latter  ganglionic  cells  may  sometimes  be  seen. 

At  the  back  part  of  the  tongue  are  little  depressions  of  the 
mucous  membrane  called  crypts  (fig.  237).  In  the  walls  of  these 


Fig.  23 1;.— Filiform  Papillae,  x 30. 
I.  Primary  papilla;  2.  Secon- 
dary papillae  on  its  summit ; 3. 
Epithelial  process  on  papilla ; 
4.  Single  process,  with  en- 
tangled loose  epithelial  cells. 


Secondary 

papillae. 

Fungiform 


FiG-  236.— Fungiform  Papilla,  x 30, 


248 


PRACTICAL  HISTOLOGY. 


[XXI. 


Epithelium 


Crypt. 

Adenoid 

tissue. 


Mucous 

glands. 


Mucous 

glands. 


Fig.  237.  —Crypt  from  Back  of  Tongue,  with  Cluster 
of  Lymphatic  Follicles. 


are  spherical  masses  of  adenoid  tissue,  and  into  some  crypts  open  the 
ducts  of  small  mucous  glands  (fig.  237). 

2.  The  mucous  glands  occur  chiefly  at  the  base  of  the  tongue 

and  along  its  edges. 
They  have  the  same 
structure  as  the  sali- 
vary glands  of  the  same 
name  (fig.  237), 
their  acini  are  lined  by 
a single  layer  of  mucous 
cells,  but  there  are  no 
demilunes.  They  are 
small  compound  tubu- 
lar mucous  glands. 

3.  The  serous  glands 
occur  only  near  the 
circum  vallate  papillae 
and  taste-bulbs.  Their 
acini  are  granular  and 
resemble  those  of  the 
parotid  gland  in  struc- 
ture (fig.  240,  d). 

4.  T.S.  Tongue  {Douhle-Stained). — Stain  a section  from  the 
posterior  part  of  the  organ,  first  with  methyl-green  and  then  with 

eosin.  Mount  in  balsam.  The 
connective  tissue  and  papillae  are 
reddish ; the  serous  glands  are  red- 
dish also,  while  the  mucous  glands 
have  a purplish-green  colour. 

5.  T.S.  Injected  Tongue  (L). — 
This  is  obtained  when  the  head  or 
whole  body  is  injected.  Observe 
the  very  vascular  muscular  portion 
and  the  papillae,  each  with  an  artery 
entering  it.  If  the  papillae  be  com- 
pound, i.e.,  beset  with  other  smaller 
secondary  papillae,  a small  capillary 
loop  passes  into  each  of  these 
secondary  papillae  ; sections  of  large 
^ o rra  T • blood-vessels  in  the  connective  tissue 

Fig.  238.— T.S.  Injected  Tongue  of  Cat.  rr 

ot  the  mucous  coat  (fig,  238).  if 
desired,  another  section  may  be  faintly  stained  with  logwood  and 
mounted  as  above. 


TASTE-BUDS. 


249 


XXI  ] 


TASTE-BUDS. 


6.  Taste-Buds,  or  the  peripheral  organs  of  taste,  occur  on  the 
fungiform  papillae  and  lateral  surface  of  the  circumvallate,  soft 
palate,  posterior  surface  of  the  epiglottis,  and  a few  amongst  the 
epithelial  cells  on  the  dorsum  and  sides  of  the  tongue.  It  is  more 
convenient,  however,  to  study  them  in  the  rabbit.  On  either  side 
of  the  posterior  part  of  the  rabbit’s  tongue  are  two  oval  patches 
with  transverse  ridges  and  intervening  furrows,  the  papillae  foliatae 

(fig-  239)- 

(i.)  Cut  out  these  parts  and  harden  them  for  fourteen  days  or 
so  in  jMiiller’s  fluid  and  then  in  spirit.  Stain  in  bulk  in  borax- 
carmine  or  haematoxylin  and  cut  in  paraffin. 

(ii.)  The  excised  organ,  with  as  little  adherent  muscle  as  possible, 
is  placed  for  one  hour  in  i per  cent,  osmic  acid,  or  pinned  on  a 
cork  and  exposed  for  the  same  time  to  the  vapour 
of  osmic  acid.  Fine  sections  are  made  across  the 
laminae,  and  stained  with  logwood  and  mounted  in 
balsam. 

(a.)  (L)  Observe  the  sections  of  the  laminae,  each 
one  with  a central  papilla  or  projection  of  connective 
tissue  (fig.  240,  1).  This  is  covered  by  many  layers 
of  stratified  epithelium. 

(1).)  Between  the  laminae  a furrow,  and  embedded 
in  the  epithelium,  on  each  side  of  this  furrow,  the 
taste-buds  (f/),  which  are  oval  in  shape,  and  composed 
of  epithelial  cells,  whose  bases  touch  the  connective 
tissue  of  the  mucous  membrane,  where  they  receive  fig.  239.— Tongue 
a branch  of  the  glosso-pharyngeal  nerve.  The  apex 
has  an  open  mouth — gustatory  pore — which  com- 
municates with  the  furrow.  The  cells  composing  the  bud  are 
arranged  somewhat  like  tlie  staves  in  a barrel. 

(c.)  Gland-ducts  open  at  the  bases  of  the  furrows,  and  if  these 
ducts  be  not  seen,  sections  of  their  acini — serous  gland — are  sure 
to  be  seen  deep  in  the  corium  (fig.  240,  d).  The  corium  has  what 
look  like  secondary  papillae  on  it,  but  they  are  really  septa  {V). 

(d.)  (H)  Study  a single  taste-bud  (80  p long  and  40  p broad) 
It  is  composed  of  two  kinds  of  elongated  epithelial  cells. 

(i.)  The  sustentacular  cells,  which  are  most  numerous.  They 
are  elongated,  flattened,  and  either  of  uniform  breadth  or  narrowed 
at  their  base.  They  form  a protective  covering  for  the  true  gusta- 
tory cells,  which  lie  between  and  within  them. 

23 


250 


PKACTICAL  HISTOLOGY. 


[xxi. 


2.)  The  gustatory  cells  consist  of  narrow  fusiform  nucleated 

cells,  whose  lower  pointed 
end  is  continuous  with  a 
branch  of  the  axial  cylinder 
of  a nerve-fibre,  while  the 
free  end  is  continued  into  a 
fine  point  or  ciliuin,  which 
projects  through  the  gus- 
tatory pore. 

If  it  be  desired  to  study 
the  mode  of  termination 
of  the  nerves  in  these 
organs,  use  the  lemon-juice 
gold  chloride  method,  with 
subsequent  exposure  of 
the  tissue  to  sunlight  in 
water  acidulated  with 
acetic  acid,  Golgi’s  rapid 
hardening  method,  or 
methylene-blue.  For  an 
elaborate  research,  with 
beautiful  plates  showing 
the  terminations  of  the 
nerves  in  the  papillae  foliatae,  see  Drasch.^ 


Fig.  240. — Papillae  Foliatae  in  the  Uahbit.  /,  V.  Pri- 
mary and  secondary  septa ; g.  Taste-huds ; n. 
Medullated  nerve  ; d.  Serous  gland  ; a.  Its  duct ; 
M.  Muscular  fibres,  x 80. 


SOFT  PALATE. 

1.  T.S.  Soft  Palate. — Harden  the  soft  palate  of  a rabbit  or  dog 
in  Muller’s  fluid,  alcohol,  or  corrosive  sublimate.  Make  transverse 
sections  by  freezing,  or  stain  in  bulk  in  borax-carmine  and  cut  in 
paraffin. 

(a.)  (L  and  H)  One  of  the  most  beautiful  methods  of  staining 
is  that  recommended  by  List,  viz.,  to  stain  with  aniline-green  and 
eosin,  and  mount  in  balsam.  Even  with  the  naked  eye  the  thick 
layer  of  mucous  glands  can  be  seen. 

{h.)  The  stratified  epithelium  and  connective  tissue  are  rosy-red, 
the  nuclei  blue.  The  glands  are  bluish,  and  are  seen  to  be  mucous 
in  character,  lined  by  a single  layer  of  mucous  cells  without  demi- 
lunes. In  the  borax-carmine  section,  the  cells  lining  the  acini  of 
the  glands  are  clear  and  transparent  and  show  no  demilunes,  a 
typical  example  of  a pure  mucous  gland. 

^ “ TJnters.  liber  d.  Pap.  fob  et  circumvall.  d.  Kaninclieiis,”  Ahhand.  d, 
math,  -pliys.  Classe  d.  K.  Sachs.  Gesell.  d.  JVissensch. . Bd.  xxiv. 


XXII.] 


TOOTH. 


?SI 


ADDITIONAL  EXERCISES. 

Glands  of  Tongue  or  Palate. — Harden  the  tongue  or  soft  palate  of  a rabbit 
in  3 per  cent,  nitric  acid  for  1-2  hours.  Wash  out  all  the  acid  and  stain  the 
.sections  with  methylene-blue.  Wash  out  the  blue  with  alcohol  until  only  the 
glands  remain  blue.  Mount  in  xylol-balsam. 

Terminations  of  Nerves  in  the  Lingual  Papillae  and  Glands. — These  may  be 
studied  by  staining  small  pieces  of  the  tongue  of  mouse  or  rat  by  Golgi’s  silver 
nitrate  method  (p.  78),  or  by  the  rapid  hardening  method  (bichromate  of  potash 
and  osmic  acid,  Lesson  XXVI.  14).  The  nerve  in  the  papillae  contains  nerve-cells 
which  are  in  connection  with  the  nerve-fibres.  The  nerve-fibres  form  a plexus 
of  fibres  outside  the  basement  membrane  of  the  serous  gland  acini — epilemmal 
plexus — and  one  within  this  membrane  amongst  the  secretory  cells — hypo- 
lemmal  plexus  {Fusari  and  Fanasd)} 


LESSON  XXII. 

TOOTH— OESOPHAGUS. 

TOOTH. 

The  chief  mass  of  a tooth  consists  of  dentine.  It  is  capped  by 
enamel,  and  the  root  or  fang  is  invested  by  a layer  of  bone,  the 
crusta  petrosa.  All  three  tissues  are  calcified,  and  contain  calcic 
phosphate.  The  enamel,  however,  is  an  epithelial  structure,  and 
consists  of  modified  and  calcified  epithelial  cells,  while  the  dentine 
and  crusta  petrosa  belong  to  the  connective  tissue  group. 

IJnsoftened  Tooth. — This  is  one  of  the  few  preparations  which 
had  better  be  bought. 

1.  Longitudinal  Section  of  a Dry  Tooth  (fig.  241). 

(a.)  (L)  Observe  the  crown  and  fang,  and,  connecting  the  two, 
the  neck. 

(/>.)  The  dentine  surrounding  the  pulp  cavity,  the  enamel 
covering  the  dentine  of  the  crown,  and  the  crusta  petrosa  or 
cement  covering  the  dentine  of  the  fang.  The  wavy  black  lines  in 
the  dentine  or  dentinal  tubules  are  really  tubules  filled  with  air, 
hence  they  appear  black.  Note  their  direction  from  the  pulp  cavity 
towards  the  outer  margin  of  the  dentine.  Quite  at  the  apex  of  the 
crown  of  the  tooth  they  run  vertically ; in  the  fang  they  run  nearly 
horizontally,  and  in  the  part  of  the  dentine  intermediate  between 
^ Archie,  ital,  de  xiv.  p.  240,  1891. 


PHACTICAL  HISTOLOGY. 


252 


[xxii. 


both  they  gradually  become  more  and  more  oblique  from  the  centre 
of  the  crown.  ^ 

Arched  or  curved  lines — incremental  lines  or  Schreger’s  lines 

— may  sometimes  be  seen  crossing 
the  course  of  the  dentine  tubules. 

(c.)  The  enamel  covering  the 
dentine  on  the  crown  of  the  tooth; 
somewhat  brownish-coloured  con- 
centric lines  may  be  seen  in  it. 

{d.)  (H)  The  enamel  consists 
of  striated  prisms,  hexagonal  when 
seen  in  transverse  section  (fig. 

243) - 

The  dentinal  tubules  lie  in  a 
homogeneous  matrix,  and  are 
wavy  tubes,  which  divide  di- 
chotomously,  and  give  off  lateral 
branches  which  anastomose  with 
other  lateral  branches  from  ad- 
jacent tubules.  At  the  outer  part 
of  the  dentine  are  irregular  inter- 
globular  spaces,  which  appear 
black  when  they  contain  air  (fig. 

244) . 

The  crusta  petrosa  consists  of 
bone — a thin  layer — composed  of 
lamellae  and  bone-corpuscles,  but 
no  Haversian  canals  (fig.  244). 

2.  Softened  Tooth. — Select  the 
brane;  P~C.  Pulp  cavity ; C.  Cement;  jaw  of  a small  mammal,  e.g.,  a cat, 
lvi^vy;N!^Z7.  and  decalcify  a short  length— | 

inch — of  the  lower  jaw  in  chromic 
acid  and  nitric  acid.  It  will  take  two  or  three  weeks  to  remove 
all  the  bone-salts,  and  the  decalcifying  fiuid  must  be  frequently 
renewed.  The  tooth  is  sufficiently  soft  to  be  cut  when  a needle 
can  be  pushed  into  it.  Make  vertical  sections  through  the  whole 
jaw  and  a tooth  in  situ. 

Stain  one  section  in  picro-carmine,  another  in  osmic  acid  (twenty- 
four  hours),  and  mount  both  in  Tarrant’s  solution  or  in  glycerine- 
jeUy. 

(a.)  (L)  Observe  the  tooth  in  the  alveolus  or  depression  of  the 
jaw  in  which  it  is  fixed.  The  enamel  has  disappeared.  The  bone 
of  the  jaw  with  its  periosteum,  lining  the  alveolus  and  forming 
there  the  periodontal  membrane  (fig.  241,  P.M.). 

(/;.)  Next  the  latter  on  the  fang  the  cement  (C),  the  dentine. 


TOOTH. 


253 


XXII.] 

pulp-cavity  and  its  contents.  If  the  section  passes  directly  through 
the  middle  of  the  tooth,  the  orifice  in  the  fang  of  the  tooth  may  be 
seen. 

(>.)  (H)  The  dentinal  tubules,  not  so  distinct  as  in  the  dry  tooth. 


Fig.  242. — Euamel 
Prisms. 


c 

Fig.  243.— Their  Hexa- 
gonal Ends. 

If  they  are  cut  obliquely,  they  appear  merely  as  tailed  dots  in  a 
homogeneous  matrix. 

[d.)  The  pulp-cavity  contains  blood-vessels  and  fine  connective 
tissue,  but  next  the  dentine  there  is  a layer  of  large  cubical  cells — 
odontoblasts — which  give  off  fine  processes  which  enter  the  den- 
tinal tubules — the  fibres  of  Tomes.  They  are  best  seen,  however, 
in  a tooth  which  has  not  yet  cut  the  gum. 

3.  Development  of  Tooth. — Without  entering  into  all  the 
details  of  the  development  of  the  teeth,  the  following  directions 
will  suffice  as  to  the  method  of  preparing  sections  so  as  to  show  the 
various  stages.  What  may  be  called  the  first  stage — that  shown  in 
fig.  245 — is  to  be  obtained  from  the  lower  jaw  of  a sheep’s  embryo 
7 cm.  in  length.  At  this  stage  only  a very  little  bony  matter  exists. 
Harden  the  whole  jaw  in  corrosive  sublimate  and  decalcify  in  dilute 
hydrochloric  acid.  Stain  in  bulk  in  borax-carmine,  embed  in 
paraffin,  and  make  T.S.  across  both  rami  of  the  jaw  and  the  tongue. 
Or  harden  and  decalcify  at  the  same  time  the  jaw  of  a foetal  kitten 
by  placing  small  pieces  containing  embryonic  teeth  in  Flemming’s 
fluid.  This  yields  excellent  results, — the  tissues  are  thereby  suffi- 
ciently differentiated  and  may  be  cut  in  paraffin. 

The  second  stage,  fig.  246,  is  obtainable  from  the  upper  jaw  of  an 
embryo  sheep  1 5 cm.  long.  It  is  treated  in  the  same  way. 

The  third  stage,  fig.  247,  is  obtained  from  the  lower  jaw  of  a dog 
six  days  old  or  thereabout. 


tubules ; c.  Granular  or  interglobular 
layer. 


254  PKACTICAL  HISTOLOGY.  [XXII, 


With  a high  power  it  is  easy  to  observe  the  structure  of  tlie 


of  a Tooth,  a.  Epithelium 
of  dental  ridge  on  lower 
jaw ; b.  Portion  of  epithe- 
lium about  to  be  modified 
into  enamel  (enamel  organ); 

c.  Beginning  of  germ  of  den- 
tine in  the  tooth  papilla ; 

d.  Lamination  of  corium 
about  to  form  tooth  sac. 


Fig.  246. — Lower  Jaw  of  Human  Foetus  at  4th 
Month,  X 40.  I.  Dental  ridge ; 2.  Stalk  of 
enamel  germ ; 3.  Enamel  organ ; a.  Peri- 
pheral cells ; h.  Germ  pulp ; c.  Cylindrical 
cells  of  enamel ; 4.  Papilla. 


several  parts,  and  to  see  the  odontoblasts  lining  what  is  to  be  the 


( Outer  layer. 
Dental  Sac.  •< 

(Inner  layer. 

Dentine. 

{Odontoblasts. 

Spherical  elements 
with  blood-vessels. 


Bone  of  the  lower  jaw.^ 


Peripheral  flat 
cells. 

Enamel  pulp. 
Enamel  cells. 


Enamel 

organ. 


Enamel. 


Fig.  247. — T.S.  Lower  Jaw  of  New-Born  Dog,  x 40.  The  dental  sac  is  shown  only  in  the 
left  side.  The  tissues  originating  from  connective  tissue  are  shown  on  the  left,  and 
those  of  epithelial  origin  on  the  right. 


pulp-cavity.  If  the  latter  happen  to  be  partially  detached,  their 


XXII.] 


CESOPHAGUS. 


255 


processes — fibres  of  Tomes — may  be  seen  partially  withdrawn  from 
the  dentinal  tubules  in  which  they  lay. 


(ESOPHAGUS. 

The  (Esophagus  consists  from  within  outwards  of — 

(i.)  Mucous  coat,  composed  of  stratified  squamous  epithelium, 
into  which  project  small  simple  papillae  from  the  corium  or  con- 
nective-tissue basis  of  the  membrane.  At  the  outer  part  of  the 
corium  is  a narrow  layer  of  smooth  muscular  fibres — muscularis 
mucosae — arranged  for  the  most  part  longitudinally. 

(2.)  Submucous  coat,  consisting  of  connective  tissue  and  the 
larger  blood-vessels  and  some  nerves.  In  those  animals  {e.g.^  dog) 
in  which  glands  occur,  the  acini  of  the 
glands  lie  in  the  submucous  coat,  so  that 
their  ducts  have  to  perforate  the  muscu- 
laris  mucosae  and  traverse  the  mucous 
membrane  before  they  open  on  the  surface 
of  the  epithelium. 

(3.)  Muscular  coat. — This  will  vary 
with  the  animal  used.  In  man  this  coat 
in  the  upper  third  of  the  oesophagus  is 
composed  of  striated  muscular  fibre,  the 
lower  two-thirds  of  smooth  muscle.  The 
outer  layer  of  fibres  runs  longitudinally, 
the  inner  circularly. 

(4.)  Fibrous  coat,  composed  of  fibrous 
tissue. 

4.  The  (Esophagus.— Cut  out  a piece 
of  the  oesophagus  of  a dog  or  cat — 2 cm. 
in  length — and  harden  it  in  equal  parts 
of  chromic  acid  (|  per  cent.)  and  spirit,  or 
Muller’s  fluid  (fourteen  days),  or  mercuric 
chloride,  and  then  in  alcohol.  Make 
transverse  sections ; stain  one  in  j3icro- 
carmine  and  mount  it  in  Tarrant’s  solu- 
tion, and  another  in  logwood  and  mount 
it  in  balsam.  Perhaps  even  more  instructive  sections  are  obtained 
from  a small  animal,  such  as  a rat,  where  the  Avhole  oesophagus  (with 
trachea)  can  be  stained  ‘4n  bulk”  in  borax-carmine.  Cut  sections 
of  both  tubes  to  show  their  structure  and  relations. 

(a.)  (L  and  H)  The  circular  tube  has  several  coats,  the  inner- 
i^ost  one  being  thrown  into  folds.  The  mucous  membrane  covered 
by  stratified  squamous  epithelium,  under  this  the  connective  tissue 


Fig.  248. — T.S.  Small  Part  of 
(Esophagus  of  Dog,  E.  Epi- 
thelium ; M.M.  Muscularis 
mucosae ; G.  Glauds  ; C.  Cir- 
cular, and  L.  External  or 
longitudinal  muscular  coat; 
F.  Fibrous  layer.  Muller’s 
fluid,  picro-carmine. 


256 


PRACTICAL  HISTOLOGY. 


[XXII. 


with  small  simple  papilloe.  In  the  deeper  part  of  the  mucous  mem- 
brane are  several  layers  of  non-striped  muscle,  the  muscMlaris 
mucosae  (248,  M,M). 

{h.)  Outside  this  is  loose  submucous  connective  tissue  with  a 
few  blood-vessels,  and  in  the  dog  and  some  other  animals  the  acini 
of  mucous  glands.  The  ducts  of  the  latter  traverse  the  coats  lying 
internal  to  the  glands,  and  open  on  the  inner  surface  by  funnel- 
shaped  openings. 

(c.)  Outside  this,  again,  is  the  muscular  coat,  which  varies  in 
the  upper  and  lower  parts  of  the  tube,  and  also  with  the  animal 
examined.  In  the  upper  part  there  is  striped  muscle,  in  the  lower 
part  two  layers  of  non-striped  muscle,  an  inner  circular  and  an  outer 
longitudinal.  Between  the  two  layers  may  be  seen  ganglionic  cells 
of  Auerbach’s  plexus. 

(d.)  Outside  all  is  the  fibrous  coat  or  adventitia,  composed  of 
coarser  connective  tissue,  with  elastic  fibres  and  blood-vessels. 

For  the  epithelial  cells  lining  the  oesophagus  see  Lesson  TV.  5. 

It  is  to  be  remembered  that  there  are  very  great  differences  as 
regards  the  presence  of  glands  in  the  oesophagus.  Some  animals 
have  a considerable  number — e.g.,  dog — and  others  very  few. 


ADDITIONAL  EXERCISE. 

5.  Other  Methods  ((Esophagus).  — Very  good  results  are  obtained  by  harden- 
ing in  absolute  alcohol  containing  methyl-green,  the  gland-cells  being  thereby 
sharply  defined  {Ruheli).^  Also  double  stain  with  borax-carmine  (extract  with 
acid-alcohol),  then  alcohol,  and  stain  again  with  iodine-green  (twenty-four 
hours),  extract  with  alcohol,  embed,  and  cut  in  paraffin.  The  mucous  mem- 
brane of  some  animals  {e.g.,  pig)  contains  lymph  follicles,  which  can  readily 
be  detected  in  a part  of  the  tube  stained  in  bulk  in  borax-carmine. 


LESSON  XXIII. 

THE  SALIVARY  GLANDS  AND  PANCREAS. 

THE  SALIVARY  GLANDS. 

All  these  glands  have  not  the  same  structure,  hence  it  is  necessary 
to  classify  them. 

Mucous  Salivary  Glands. — The  sub-maxillary  and  sub-lingual 
glands  of  the  dog  and  sub-lingUal  of  guinea-pig. 

^ Zeits.f.  mik.  Anat.,  vii.  p.  224,  1890. 


XXIII.]  THE  SALIVARY  GLANDS.  257 

Serous  Salivary  Glands. — The  parotid  of  man  and  mammals, 
and  the  sub-maxillary  of  the  rabbit. 

Mixed  or  Muco-Salivary. — The  human  sub-maxillary,  retro- 
lingual  of  the  dog,  or  sub-maxillary  of  the  guinea-pig. 

The  salivary  glands  are  compound  tubular  glands,  t.e.,  the  duct  is 
branched,  while  the  acini  or  alveoli — the  true  secretory  parts  of 
the  glands — are  tubular  in  form.  Each  gland  consists  of  lobes, 
held  together  by  connective  tissue,  which  forms  a capsule  for  the 
whole  gland  and  gives  septa  to  enclose  the  lobes  and  lobules. 
Each  lobe  in  turn  is  made  up  of  numerous  smaller  lobules  also  held 
together  by  connective  tissue,  which  carries  the  blood-vessels, 
nerves,  lymphatics,  and  larger  ducts.  From  mutual  pressure  the 
lobes  and  lobules  are  usually  polygonal  in  shape.  The  main  duct 
is  made  up  by  the  convergence  of  ducts  from  the  lobes — lobar 
ducts — while  from  each  lobule  there  is  a duct — lobular  ducts, 
which  unite  to  form  lobar  ducts.  Each  lobule  is  made  up  of  a 
number  of  alveoli  or  acini.  Each  alveolus,  which  has  a closed 
extremity,  leads  into  or  discharges  its  secretion  into  a fine  duct  or 
ductule,  and  these  ductules  by  their  union  form  the  intralobular 
ductSo  Practically  the  arrangement  of  the  ducts  is  the  same  in  all 
this  set  of  glands ; the  difierences  in  structure  are  in  the  alveoli. 
The  alveoli  consist  of  a basement  membrane,  which  by  appro- 
priate means  can  be  shown  to  consist  of  branched  cells  forming  a 
reticulated  or  basket-like  membrane.  This  is  lined  internally  by 
the  secretory  epithelium,  leaving  a larger  or  smaller  lumen  in  the 
centre,  which  leads  into  a fine  duct  or  ductule  by  means  of  a narrow 
juncMonal  piece  or  intermediary  or  ductiile^  in  which  the  epi- 
thelium is  somewhat  flattened.  Usually  several  alveoli  open  into 
one  intermediary  tubule  or  ductule.  The  ducts  with  a fibrous  wall 
are  lined  by  a single  layer  of  columnar  epithelium,  which  is  striated 
or  “rodded”  in  its  outer  part,  and  granular  towards  the  lumen  of 
the  tube  (fig.  249) ; a little  inwards  from  the  centre  of  each  cell  is 
a nucleus. 

In  mucous  glands  the  acini  (35  ju.  in  diameter)  are  lined  by  a 
layer  of  polyhedral  clear  cells,  whose  broader  bases  rest  on  the 
basement  membrane,  while  their  apices  abut  on  the  lumen,  which 
is  small  (fig.  249).  Usually  in  a transverse  section  of  an  acinus 
five  or  six  cells  are  seen.  The  appearance  of  these  cells  varies 
according  as  a gland  is  at  rest  or  in  a state  of  activity,  i,e.^  whether 
the  gland  is  “loaded”  or  “charged”  (resting  phase),  or  “unloaded” 
or  “ discharged  ” (active  phase).  In  a resting  gland  the  mucous 
cells  are  clear,  for  the  most  part,  while  at  the  outer  part  of  the  cell 
is  a flattened  nucleus  surrounded  by  a very  small  quantity  of 
granular  protoplasm.  The  clear  part  is  traversed  by  a network  of 
fibrils,  which  includes  in  its  meshes  mucigen.  The  granular  matter 

R 


258  PRACTICAL  HISTOLOGY.  [XXIII. 

and  nuclei  stain  readily  with  the  ordinary  dyes,  while  the  clear 
part  does  not  do  so. 

In  some  mucous  glands,  e.g,  dog,  but  not  in  all  mucous  glands, 
here  and  there  between  the  bases  of  some  of  these  cells,  and  the 
basement  membrane,  are  groups  of  smal],  granular,  nucleated  cells, 
the  group  having  a somewhat  crescentic  shape  ; they  are  called 
demilunes  or  crescents  of  Gianuzzi  (fig.  250).  They  stain  readily 
with  dyes,  and  are  darkened  by  osmic  acid,  and  contain  two  or 
more  nuclei. 

In  the  discharged  or  active  gland,  the  acini  are  smaller,  the 
lumen  wider,  the  clear  part  of  the  cell  diminished  in  volume,  while 
the  outer  part  of  the  cell  is  wider,  and  appears  to  have  encroached 
on  the  clear  part.  The  nucleus  is  usually  spherical,  and  placed 
nearer  the  centre  of  the  cell. 

In  serous  or  albuminous  glands  the  chief  differences  are  in  the 
cells  lining  the  alveoli.  In  serous  alveoli  there  is  but  one  layer  of 
cells,  and  nothing  corresponding  to  the  demilunes.  The  cells  are 
somewhat  smaller  than  mucous  cells ; they  are  more  granular,  and 
stain  more  uniformly  with  dyes.  The  nucleus  is  spherical,  and 
placed  nearer  the  centre  of  the  cell.  The  differences  between 
active  and  passive  phases  are  not  so  marked  as  in  mucous  acini. 

During  activity  the  cells  become  smaller,  and  the  ‘^granules’’ 
disappear  from  the  outer  part  of  the  cell ; the  cells  become  more 
sharply  defined,  while  the  nuclei  are  large  and  spherical. 

N.B. — In  all  cases  examine  the  acini  of  the  glands  in  the  fresh 
condition. 

A.  Mucous  Salivary  Glands. — These  must  be  prepared  in 
several  ways.  ^ 

Methods. — (i.)  For  the  general  structure  of  salivary  glands : — 

Harden  small  piece ; for  2 or  3 days  in  the  following  mixture  : — 
3 parts  90  per  cent,  alcohol  and  2 parts  .5  per  cent,  chromic 
acid.  To  see  the  finer  points — after  staining — mount  the  sections 
in  glycerine  [Langley). 

(ii.)  Small  pieces  of  a perfectly  fresh  dog’s  sub-maxillary  gland 
are  placed  for  an  hour  in  75  per  cent,  alcohol,  then  for  five  hours 
in  absolute  alcohol,  which  is  then  changed,  and  the  hardening  is 
completed  in  fresh  absolute  alcohol  in  twenty-four  hours.  Sections 
of  the  unstained  gland  are  apt  to  fall  to  pieces,  although  the  small 
pieces  show  the  structure  sufficiently  well.  A part  of  the  alcohol- 
hardened  gland  should  be  stained  “ in  bulk  ” in  borax-carmine,  and 
cut  in  paraffin.  In  this  way  the  relative  position  of  the  parts  is 
retained. 

(iii.)  Harden  very  small  pieces  in  i per  cent,  osmic  acid 
(24  hours) ; wash  thoroughly,  and  complete  the  hardening  in 
alcohol. 


xxiil]  the  salivary  glands.  259 

(iv.)  Harden  other  pieces  in  Flemming’s  mixture,  and  stain  ‘‘in 
bulk  ” in  borax-carmine  as  in  (ii.). 

(v.)  Heidenhain’s  Method.  ^ — This  method  is  also  applicable 
to  the  pancreas.  Small  pieces  of  glands — sub-maxillary  of 
guinea-pig,  dog,  and  cat — hardened  in  alcohol  (ii.)  are  })laced  in  10 
cc.  of  0.5-1  per  cent,  watery  solution  of  hsematoxylin  (6-8  hours); 
and  then  for  an  equal  period  in  0.5-1  per  cent,  potassic  bichromate, 
or  I per  cent,  watery  solution  of  alum,  or  stain  with  .3  per  cent, 
hsematoxylin  (distilled  water  to  he  used),  and  differentiate  with  i 
per  cent,  neutral  chromate  of  potash,  which  forms  a steel-grey 
compound  with  hsematoxylin  (p.  70).  The  stain  does  best  with 
objects  hardened  in  alcohol  or  picric  acid.  The  pieces  are  quite 
black  when  removed  from  the  second  fluid.  In  this  method,  the 
union  of  the  reagents  takes  place  in  the  tissue  itself.  The  nuclei 
are  bluish-black,  the  cell-substance  a steel-grey,  while  the  demilunes 
stand  out  distinctly.  This  is  an  excellent  method  for  these  glands. 
Cut  in  paraffin. 

(vi.)  Mucous  glands  harden  well  in  picric  acid. 

My  experience  leads  me  to  believe  that  in  studying  a mucous 
salivary  gland,  it  is  best  to  begin  with  one  whose  acini  contain  only 
mucous  cells  and  no  demilunes.  Such  glands  are  the  sub-lingual 
of  a guinea-pig  and  sub-maxillary  gland  of  the  mole.  Then  proceed 
to  the  sub-maxillary  of  a dog,  which  has  demilunes  at  intervals, 
and  lastly,  take  the  acini  of  a cat’s  sub-maxillary,  where  the  demi- 
lunes form  a nearly  complete  layer  outside  the  true  mucous  cells. 

In  order  to  obtain  a general  view  of  the  origin  of  the  ducts  from 
the  acini  of  the  lobules,  and  the  union  of  small  lobular  ducts  to 
form  larger  ducts,  it  is  well  to  examine  a preparation  of  the  salivary 
glands  of  the  cockroach,  which  can  be  removed  en  masse  (p.  265). 

1.  Sub-Lingnal  Gland  of  Guinea-Pig. — This  shows  acini  lined  with 
mucous  cells  without  demilunes  (L  and  H).  Hote  the  acini,  each  lined 
by  a single  layer  of  clear  transparent  mucous  cells,  resting  directly 
upon  a basement  membrane  without  the  intervention  of  any  demi- 
lunes. In  the  arrangement  of  capsule,  septa,  and  ducts,  it  resembles 
the  salivary  glands  of  the  dog.  The  gland  is  like  flg.  249  without 
the  demilunes. 

2.  Sub-Maxillary  Gland  of  Dog. — (a.)  (L)  Observe  the  capsule, 
which  sends  off  thin  connective-tissue  septa  into  the  gland,  mapping 
it  out  in  polygonal  lobes,  which  are  further  subdivided  by  liner 
septa  into  lobules.  In  the  larger  septa,  sections  of  blood-vessels  and 
gland-ducts  (fig.  249). 

(h.)  Within  each  lobule  aggregations  of  acini  or  alveoli,  which 
make  up  the  smaller  lobules.  The  shape  of  the  lobule  depends  on 
the  way  it  has  been  cut.  Branches  of  the  finer  gland-ducts — few — 
^ Archiv /.  mile.  AnaL,  1884,  p.  468,  and  1886,  p.  383. 


26o 


PRACTICAL  HISTOLOGY 


[XXIII. 


Fig.  249. 


Small  Lobule  of  a Sub-Maxillary  Gland,  Dog. 
L.  Lobule  ; D.  Duct.  Osmic  acid. 


between  the  lobules.  Some  of  the  acini  appear  to  be  crowded  with 
cells  inside  the  basement  membrane.  The  gland-ducts  have  a 
distinct  lumen. 

(c.)  (H)  Although  numerous  acini  are  visible,  only  those  that 

have  been  cut  across 
so  as  to  show  the 
lumen  are  satisfactory; 
the  others  appear 
merely  to  be  filled 
with  cells,  and  vary 
much  in  size,  accord- 
ing to  the  plane  of 
section  through  the 
alveolus.  Each  aci- 
nus has  a clear,  trans- 
parent basement  mem- 
brane, and  inside,  and 
on  it,  is  arranged  the 
secretory  epithelium. 
{d.)  The  mucous  cells  form  a single  layer,  and  are  large,  clear, 
cubical  cells.  Each  has  a nucleus  which  is  usually  flattened  and 
placed  near  the  attached  end  of  the  cell.  In  borax-carmine  pre- 
parations, the  nucleus,  surrounded  by  granular  protoplasm,  is  stained 
red,  while  the  rest  of  the  cell  appears  clear,  and  traversed,  it  may 

be,  by  fine  threads  or 
fibrils.  In  reality,  the 
cell-substance  consists  of 
a network  containing  a 
clear  substance  — muci- 
gen. 

(c.)  The  demilunes  lie 
singly  next  the  basement 
membranes.  Two  or 
three  may  bd  seen  in  the^ 
section  of  each  alveolus 
as  more  granular  deeply- 
stained  bodies,  sometimes 
with  two  nuclei.  As 
their  name  indicates, 
they  are  somewhat  half- 
moon shaped,  but  they 
send  processes  between  the  mucous  cells.  As  they  are  deeply 
stained  by  pigments  and  also  darkened  by  osmic  acid,  their  shape 
and  distribution  are  readily  recognised. 

(/.)  The  lumen  of  each  acinus  is  a small  more  or  less  regular 


Fig.  250.— Sub-Maxillary  Gland,  Dog,  showing  Duct 
Communicating  with  an  Alveolus  by  a Narrow 
Ductule.  The  alveoli  with  mucous  cells  and  dense 
demilunes.  Osmic  acid  and  hsematoxylin,  x 300. 


xxiil] 


THE  SALIVARY  GLANDS. 


261 


space  in  the  centre  of  the  acinus,  hut  many  acini  may  he  so  divided 
as  not  to  show  it.  It  is  difficult  to  find  the  connection  between  the 
lumen  of  an  acinus  and  a duct  (fig.  250). 

(g.)  Duct. — This  is  hest  studied  in  a transverse  section  of  one  of 
the  finer  ducts  lying  within  the  lobules  or  the 
larger  ones  in  the  septa  (fig.  251).  The  wall 
consists  of  circularly  disposed  connective  tissue, 
and  is  lined  by  a single  layer  of  tall,  narrow, 
cylindrical  epithelium.  The  outer  part  of  each 
cell  is  distinctly  striated  or  “rodded/'  and  the 
spherical  nucleus  is  placed  about  the  middle  of 
the  cell. 

N.B, — The  student  should  compare  with  this  fiq.  251-— T-S.  Salivary 
a section  of  a gland  hardened  in  osmic  acid  or  the  “ Rodded  ” Epi- 

riemming’s  fluid,  or  one  stained  by  Heidenhain’s  theimm  Lining  it, 

method. 

3o  Sub-Maxillary  Gland  of  Cat. — In  some  of  the  acini  there 
may  be  a nearly  complete  layer  of  demilune  cells  between  the  base- 
ment membrane  of  the  acinus  and  the  lining  layer  of  mucous  cells. 

B.  Serous  Salivary  Glands — Methods. — Use  the  parotid  of  any 
mammal — rabbit,  cat,  or  dog — or  the  sub-maxillary  gland  of  a 
rabbit.  Harden  small  parts  of  the  gland  in  the  same  way  and  by 
the  same  means  as  for  mucous  glands.  A saturated  watery  solution 
of  mercuric  chloride  is  to  be  pre- 
ferred to  picric  acid  for  serous 
glands.  I find  that  Tlemming’s 
mixture  is  specially  good  for  the 
sub-maxillary  gland  of  the  rabbit. 

Sections  are  made  and  stained — the 
hardened  gland  stained  “in  bulk’^ 

— just  as  for  mucous  glands. 

4.  T.S.  Parotid  Gland.  — (a.) 

(L)  Observe  the  capsule,  septa, 
lobes,  and  lobules  as  in  the  mucous 
glands,  but  the  alveoli  or  acini  are 
smaller.  More  sections  of  gland- 
ducts  will  also  be  seen. 

{h.)  (H)  Observe  an  acinus.  It 
is  lined  by  a layer  of  polyhedral 
cells,  leaving  a very  small  lumen.  The  cells  are  very  granular,  with 
a spheroidal  nucleus  placed  near  the  centre  of  the  cell  (fig.  252). 
Numerous  sections  of  ducts,  some  cut  transversely,  others  iongitudi- 
nally.  They  are  like  those  of  mucous  glands. 

5.  Fresh  Serous  Gland. — Tease  a fragment  of  a parotid  gland  in 
normal  saline,  and  observe  how  the  cells  are  crowded  with  granules. 


Fig.  252.— Resting  Serous  Gland,  Rabbit. 
Alcohol  and  carmine. 


262  PRACTICAL  HISTOLOGY.  [XXIIl. 

C.  Muco-Salivary  Glands,  e.y.,  sub-maxillary  of  man  or  retro- 
lingual  of  the  dog,  are  treated  as  the  other  salivary  glands. 

6.  Human  Sub-Maxillary  Gland. — (L  and  H)  Observe  that  some 
of  the  acini  are  like  those  of  mucous  glands,  and  others  like  serous 
acini,  while  some  of  the  acini  contain  both  mucous  and  serous 

cells.  Acini,  serous  and 
mucous,  may  be  found 
lying  side  by  side  (fig. 
253)- 

7.  Dog’s  Sub-Maxillary 
Gland  (Double-Staining). 
— (i.)  Stain  alcohol-hard- 
ened sections,  first  in  a 
watery  solution  of  aniline 
green  and  subsequently  in 
eosin.  An  easier  plan  is 
to  stain  first  in  aniline 
green,  rapidly  dehydrate 
the  section  in  alcohol, 
taking  care  that  all  the 
green  is  not  washed  out 
of  the  gland-cells,  and  clarify  with  clove- oil  in  which  is  dissolved 
some  eosin.  Mount  in  balsam.  The  mucous  cells  are  green,  the 
demilunes  pinkish,  and  the  nuclei  generally  are  green.  The  cells 
of  the  ducts  are  green,  and  the  interlobular  connective  tissue 
pinkish, 

(ii. ) Stain  sections  in  aniline  blue,  to  which  is  added  a saturated 
watery  solution  of  picric  acid.  Mount  in  balsam.  The  cells  of  the 
acini  are  blue,  while  the  ducts  are  yellowish-green. 

(iii.)  Schiefiferdecker’s  method  is  also  to  be  recommended.  Add 
a few  drops  of  a 5 per  cent,  alkaline  alcoholic  solution  of  eosin  to 
a watch-glassful  of  alcohol.  Allow  the  sections  to  stain  in  this  for 
half  an  hour  or  so,  and  place  them  for  a few  minutes  in  i per  cent, 
watery  solution  of  aniline  green,  and  mount  in  balsam. 

(iv.)  The  sub-maxillary  gland  of  a dog  or  guinea-pig  hardened  in 
picric  acid  or  HgClg,  if  stained  with  aniline  blue  and  safranin, 
shows  the  demilunes  red  and  the  mucous  cells  blue  in  balsam 
preparations. 


THE  PANCKEAS. 

The  pancreas  is  a compound  tubular  gland,  and  resembles  the 
serous  salivary  glands  in  the  arrangement  of  its  capsule,  lobes, 
lobules,  and  duct,  with  its  branches.  The  epithelium  of  its  ducts, 
however,  is  not  so  distinctly  striated,  and  in  a section,  as  a rule, 


XXIII.] 


THE  PANCREAS. 


263 


not  many  ducts  are  visible.  Curious  groups  of  cells,  “inter-tubular 
cell  clumps,”  each  supplied  by  a glomerulus-like  tuft  of  capillaries, 
lie  in  the  interlobular  septa  or  amongst  the  acini.  The  alveoli, 
tubular  or  flask-shaped,  with  a very  small  lumen,  consist  of  a base- 
ment membrane  lined  by  a single  layer  of  columnar  or  pyramidal 
cells,  each  showing  two  zones ; an  outer  zone  nearly  homogeneous, 
and  staining  with  logwood  and  some  other  dyes,  and  an  inner  zone 
crowded  with  “ granules. The  spherical  nucleus  lies  about  tlie 
middle  of  the  cell.  Sometimes  sections  of  Pacinian  corpuscles  and 
groups  of  nerve-cells  are  found  in  the  pancreas. 

This  gland,  like  other  glands,  should  be  examined  in  different 
phases  of  physiological  activity.  One,  the  active  state  (“dis- 
charged ”),  when  the  gland  is  removed  from  the  body  (rabbit)  two 
or  three  or  four  hours  after  a full  meal ; and  the  other,  the  passive, 
or  better,  the  resting  state  (“charged”  or  “loaded”),  when  the 
pancreas  is  not  secreting  actively,  which  can  be  secured  by  allowing 
an  animal  to  fast  for  fourteen  hours  (dog  or  rabbit).  The  human 
pancreas  is  rarely  satisfactory. 

Methods. — (i.)  One  of  the  best  methods  of  fixing  the  pancreas  is 
I per  cent,  osmic  acid  or  Flemming’s  mixture  (24  hours).  It  is 
then  to  be  thoroughly  washed  and  hardened,  first  in  75  per  cent., 
and  afterwards  in  absolute  alcohol.  In  such  a preparation  the 
“ granules  ” are  usually 
well  preserved,  and  they 
stain  deeply  with  saf- 
ranin. 

(ii.)  A piece  hardened 
in  absolute  alcohol  and 
stained  in  bulk  in  borax- 
carmine  or  Heidenhain’s 
logwood  (p.  70),  and 

afterwards  cut  in  paraf- 
fin, shows  well  the 
general  arrangement. 

(hi.)  Fix  a small  piece 
in  corrosive  sublimate, 

and,  after  the  usual  pre-  Fiq.  254.— T.S.  Pancreas,  Bog.  A.  Acinus;  C.  Capsule; 
cautions,  stain  the  sec-  Buct.  Corrosive  sublimate  and  picro-carmine, 

tions  with  picro-carmine. 

]\Iount  the  osmic  acid  sections  in  glycerine,  and  the  stained  ones 
in  Far  rant’s  solution  or  balsam. 

8.  Resting  Pancreas. — (a  ) (L)  Observe  the  capsule  (thinner), 
septa  (thinner),  lobes,  and  lobules,  as  in  salivary  glands.  This 
arrangement  is  well  seen  in  the  carmine  specimen  (fig.  254,  C). 

(^.)(H)  In  the  osmic  acid  preparation  observe  the  alveoli  (fig. 


264 


PRACTICAL  HISTOLOGY. 


[XXIII. 


254,  A),  with  a basement  membrane  lined  by  a single  layer  of 
columnar  cells,  tapering  somewhat  at  their  central  ends,  leaving  a 
small  irregular  central  lumen. 

(c.)  In  each  cell  a crowd  of  dark  “granules,”  occupying  about 
the  inner  two-thirds  of  each  cell,  while  the  outer  third  or  zone  is 
comparatively  homogeneous  and  free  from  granules  (Hg.  254).  The 
nuclei  of  the  cells  are  apt  to  be  obscured  by  the  presence  of  the 
granules.  In  the  borax-carmine  preparation  the  nuclei  and  granules 
are  stained  red  ; the  outer  zone,  about  one-third,  is  also  stained  red, 
the  inner  two-thirds  being  either  not  stained  or  only  faintly  so,  and 
granular,  but  the  granules  are  not  so  sharply  defined  as  in  the  osmic 
acid  preparation. 

{d,)  The  ducts  (few),  lined  by  a single  layer  of  columnar  epithe- 
lium, with  very  faint  longitudinal  striation  (fig.  254,  D). 

9.  Active  Gland  (H).  — The  cells  of  the  alveoli  are  less  granular, 
so  that  each  cell  shows  an  outer  zone  with  no  granules,  occupy- 
ing about  one-half  of  the  cell,  and  an  inner  granular  zone  with 
granules,  which,  however,  are  not  nearly  so  numerous  as  in  the 
resting  alveoli.  The  nucleus  (sometimes  with 
a nucleolus  and  accessory  nucleoli)  is  distinct,  and 
near  the  centre  of  the  cell.  It  is  to  be  noted, 
however,  that  all  the  alveoli  are  not  in  the  same 
phase. 

10.  Fresh  Pancreas.  — A very  good  view  of  the 
granular  character  of  the  inner  zone  of  the  pan- 
creatic cells  is  obtained  by  examining  a piece  of 
fresh  pancreas — e.g,  ox — teased  in  normal  saline. 
^^^AcnirS'^a  Fresh  It  is  easy  to  observe  the  difference  between  the 

Pancreas  outer  homogeneous  zone  and  the  inner  granular 

one.  Many  of  the  granules  are  liberated  in  the 

process  (fig.  255). 

11.  Injected  Pancreas  (L). — Study  a section  with  its  blood- 
vessels injected.  It  is  very  vascular,  and  in  the  inter-tubular  cell 
clumps  are  groups  of  capillaries.  It  is  best  injected  with  a Berlin- 
blue  gelatine  mass  from  the  thoracic  aorta. 


ADDITIONAL  EXERCISES. 

12.  Active  Mucous  Gland. — The  student  should  examine  a section  of  a sub- 
maxillary gland  (dog),  which  has  been  in  action  for  several  hours.  He  cannot 
without  a license  prepare  such  a gland  for  himself.  It  is  prepared,  however, 
by  stimulating  at  intervals  for  several  hours  the  chorda  tympani  of  a dog.  In 
this  way  the  gland  is  kept  secreting,  or  it  may  be  stimulated  by  injecting 


XXIII.l 


THE  PANCREAS. 


265 


pilocarpin.  It  is  then  removed  from  the  body  and  hardened  in  one  of  the 
ways  stated  on  p.  258.  It  is  essential  that  the  active  and  non-active  glands 
be  hardened  in  the  same  way,  so  as  to  show  that  such  differences  as  exist  are 
not  due  to  the  method  of  hardening. 

(H)  Observe  that  the  mucous  cells  are  not  so  clear  as  in  the  passive  gland, 
but  are  more  granular  and  smaller,  while  in  stained  sections  part  of  their  cell- 
substance  is  stained  by  the  pigments,  and  thus  there  is  less  difference  between 
the  demilunes  and  the  mucous  cells.  The  nuclei  become  more  spheroidal.  All 
the  acini  in  the  section  are  not  necessarily  in  the  same  phase  of  activity,  so 
that  the  appearances  in  any  two  acini  may  not  be  identical. 

13.  Isolated  Mucous  and  Demilune  Cells. — (i.)  Place  fragments  of  afresh 
dog’s  sub-maxillary  gland  in  5 per  cent,  ammonium  chromate  (4-6  days). 
Tease  a small  piece  in  the  same  fluid.  Note  the  isolated  mucous  cells,  each 
with  its  fibrillar  network,  spherical  nucleus  embedded  in  protoplasm,  and 
what  was  the  attached  end  of  the  cell  prolonged  into  a process. 

(ii. ) The  cells,  membranes,  &c. , are  readily  isolated  in  33  per  cent,  caustic 
potash. 

14.  Mucous  Granules. — The  mucous  granules  are  readily  seen  by  ‘‘fixing’* 
a small  piece  of  the  gland  with  the  vapour  of  osmic  acid  {Langley),^ 

15.  Salivary  Glands  of  Cockroach.  — It  is  better  to  use  the  species  PerU 
jflaneta  americana.  Kill  the  animal  with  chloroform.  Pin  it  out  on  its  back 
on  a cork  plate  and  make  the  dissection  in  normal  saline.  On  cutting  open 
the  thorax  longitudinally  one  sees  the  intestinal  tract,  and  on  each  side  of 
this,  lying  on  the  wall  of  the  latter,  aie  the  flattened  salivary  glands.  They 
can  readily  be  removed.  They  may  be  examined  fresh,  or  stained  in  picro- 
carmine,  or  exposed  to  the  vapour  of  osmic  acid,  and  mounted  in  glycerine. 
They  show  well  the  general  arrangenient  of  ducts  and  lobules.  Each  duct  is 
lined  by  a spiral  chitinous  fibre  like  the  tracheae. 

16.  Terminations  of  Nerves  in  Serous  Glands. — The  rapid  hardening 
method  of  Golgi  (Lesson  XXX.)  as  directed  for  the  pancreas  has  been  used  by 
Fusari  and  Panasci  ^ for  the  nerve  terminations  in  the  serous  glands  of  the 
tongue  of  the  rat,  rabbit,  and  cat.  The  terminal  fibrils  form  an  epilemmal 
plexus,  i,e.,  outside  the  basement  membrane,  and  other  fibrils  pass  between  the 
gland  cells,  ^.e.,  are  hypolemmal.  Fusari’s  paper  is  accompanied  by  a plate. 

17.  Gland-Ducts. — Sometimes  on  using  Golgi’s  method  one  gets  the  lumen 
of  the  ducts  black.  If  this  happens,  then  an  excellent  view  is  obtained  of 
their  course  and  connections. 

18.  Fresh  Pancreas. — In  the  rat  and  rabbit  the  pancreas  is  spread  out  in 
lobules  in  the  mesentery,  and  if  a piece  of  this  containing  a thin  part  of  the 
pancreas  be  stretched  on  a ring  of  cork  the  granules  can  be  seen  in  the  fresh 
alveolar  cells.  Osmic  acid  does  not  alter  the  granules  much,  but  alcohol  does. 

19.  Changes  in  Pancreas  Cells  may  be  seen  in  frogs — one  fed  say  three  or 
four  days  previously,  and  the  other  a few  hours  before  it  is  required. 

20.  Kodded  Structure  in  Cells. — Macerate  a small  piece  of  fresh  pancreas  in 
5 per  cent,  ammonium  chromate  for  2-3  days  ; tease  and  examine  in  the  same 
fluid.  The  outer  part  of  some  of  the  cells  will  be  found  to  be  “ rodded.” 

21.  Outer  and  Inner  Zones  of  Pancreas  Cells. — {a.)  Use  the  pancreas  of  a 
starving  animal.  To  stain  the  outer  zone,  use  ammoniacal  or  borax-carmine, 
and  for  the  granules  of  the  inner  zone  stain  a section  (fixed  on  a slide)  with 
methyl-green-fuchsin-S  solution  made  by  mixing  methyl-green  (i  per  cent.) 
60  cc.  with  fuchsin-S  solution  ( i per  cent. ) 20  cc.  Stain  for  lo  minutes.  Wash 
quickly  in  water.  Mount  in  balsam.  The  granules  are  red,  and  the  outer 
zone  clear.  [Fuchsin-S  is  acid-fuchsin.  ] For  preparations  from  Flemming’s 

^ Journal  of  Physiology^  vol.  x.  p.  433. 

2 Archiv,  ital.  de  Biol.^  xiv.  p.  240,  1891, 


24 


266 


PRACTICAL  HISTOLOGY. 


[xxiv. 


fluid,  stain  with  safranin  and  wash  with  alcohol  containing  picric  acid.  The 
safranin  stains  the  granules. 

(&. ) A pancreas  fixed  in  Flemming’s  fluid  or  sublimate  is  beautifully  stained 
by  means  of  the  following  modification  by  Oppel  of  Biondi’s  fluid : — 


The  granules  are  red  and  the  nuclei  green. 

22.  Terminations  of  Nerves  in  Pancreas. — By  means  of  Golgi’s  rapid 
hardening  method  (Lesson  XXX.) — i.e.,  osmico-bichromate  fluid  and  then 
silver  nitrate — Ramon  y Cayal  and  Sala,^  and  more  recently  Erik  Muller, ^ 
have  traced  the  terminations  of  nerve-fibres  in  the  pancreas  (dog,  rabbit). 
Numerous  nerve-fibres  enter  the  pancreas  along  its  ducts,  and  terminate  in  a 
rich  plexus  of  fibrils  around  the  individual  acini.  The  nerve-fibres  which 
surround  the  acini  are  derived  from  two  sources,  some  fibre  fibrils  are  branches 
of  Remak’s  fibres,  and  others  are  processes  of  very  characteristic  cells,  which 
R.  y Cayal  calls  ‘Wisceral  sympathetic  ganglion  cells.”  These  cells  send  off 
processes  which  are  arranged  as  part  of  the  ‘‘peri-acinous”  plexus,  and  some 
of  them  penetrate  between  the  gland-cells.  Muller  finds  that  the  fibrils  lie  in 
relation  with  the  secretory  cells. 


The  walls  of  the  stomach,  like  the  intestine,  are  composed  of  four 
coats,  named  from  within  outwards — 

(i.)  Mucous  coat,  i.e.,  the  glandular  coat. 

(2.)  Submucous,  composed  of  loosely-arranged  connective 
tissue,  the  larger  blood-vessels,  lymphatics,  and  nerves. 
(3.)  Muscular,  composed  of  three  layers  of  non-striped  muscle 
— (a.)  longitudinal,  (b.)  circular,  (c.)  oblique.  In  some 
situations  only  two  layers  exist. 

(4.)  Serous,  from  the  peritoneum. 

The  mucous  coat  is  lined  by  a single  layer  of  tall,  narrow,  cylin- 
drical mucous  cells — in  reality  mucus-secreting  goblet-cells — for 
they  have  open  mouths  and  contain  mucigen.  The  cardiac  portion 
of  the  mucous  membrane  is  composed  of  tubular  glands — fundus 
glands — placed  side  by  side.  Several  gland-tubes  may  open  into 

^ Terminacion  de  los  nervios  y tuhos  glandulares  del  pancreas  de  los  verte- 
hrados^  Barcelona,  1891. 

2 Archiv /.  mik,  Anat^  xl.  p.  405,  1892. 


Methyl -green  (i  per  cent.) 
Eosin(i  percent.)  • 
Acid-fuchsin  (i  per  cent.) 
Absolute  alcohol  o 


120  cc. 


LESSOR  XXIY. 


THE  STOMACH. 


XXIV.] 


THE  STOMACH. 


267 


one  common  duct.  The  duct  is  short,  and  is  lined  by  cells  like 
those  covering  the  surface  of  the  stomach.  The  secretory  part  of 
each  gland  is  lined  throughout  by  a layer  of  polyhedral  or  short 
columnar,  granular,  nucleated  cells,  called  chief,  principal,  inner, 
or  adelomorphous  cells.  At  intervals  between  these  and  the  base- 
ment membrane  of  the  gland  are  large,  ovoid,  conspicuous,  granular 
cells — outer,  parietal,  delomorphous,  or  oxyntic.  The  lumen  of 
the  gland  is  small  and  ill-defined. 

The  pyloric  mucous  membrane  is  beset  with  pyloric  glands, 
which  have  a long  duct  or  neck,  and  are  usually  branched  at  their 
lower  ends.  The  duct  is  lined  by  a layer  of  cells  like  those  lining 
the  stomach,  and  the  secretory  part  by  a single  layer  of  short,  finely 
granular,  short  columnar  cells.  The  lumen  is  well-defined.  There 
is  more  connective  tissue  between  the  glands  than  in  the  cardiac 
portion.  Masses  of  adenoid  tissue  are  not  unfrequently  seen 
between  the  bases  of  the  pyloric  glands. 

Methods. — (i.)  Select  a cat  or  dog  that  has  hungered  for  two 
days.  Kill  the  animal,  open  the  stomach,  and  place  small  parts  of 
the  cardiac  and  pyloric  ends  (all  the  coats)  in  equal  parts  of  chromic 
acid  (I  per  cent.)  and  spirit  (7-10  days).  Change  this  fluid  within 
twelve  hours.  Complete  the  hardening  in  alcohol. 

, (ii.)  Place  small  pieces  of  the  cardiac  and  pyloric  mucous  mem- 
brane (I  inch  cubes)  in  i per  cent,  osmic  acid  (24  hours).  Wash 
well  and  harden  in  alcohol. 

(iii.)  Fix  pieces  of  the  pyloric  and  cardiac  mucous  membrane 
in  mercuric  chloride  (2-3  hours).  Take  care  to  remove  all  the  salt 
by  prolonged  washing  in  alcohol.  Perhaps  this  is  one  of  the  best 
methods  to  use. 

(iv.)  Absolute  alcohol  is  also  a good  hardening  medium. 

To  bring  out  all  the  chief  characters  of  the  glands,  some  sections 
are  to  be  stained  in  logwood  and  mounted  in  balsam.  The  sections 
may  be  cut  by  freezing  or  in  paraffin,  after  staining  in  bulk.  Stain 
others  in  picro-carmine,  and  mount  in  Tarrant’s  solution ; others  in 
dilute  carmine  (24  hours).  Stain  others  with  i p.c.  watery 
solution  of  aniline-blue  (Nicholson’s  No.  i)  for  twenty  minutes. 
Wash  in  glycerine  and  water,  and  mount  in  Tarrant’s  solution. 
Tor  other  methods  (p.  271). 

1.  V.S.  Cardiac  End. — (a.)  (L)  Observe  the  relations,  relative 
thickness,  and  structure  of  the  several  coats.  The  mucous  coat, 
with  its  gastric  or  cardiac  glands,  or  glands  of  the  fundus,  set 
vertically  like  so  many  tubes  in  a rack  (fig.  256).  The  glands  are 
simple  tubular  glands,  and  some  of  them  have  several  secretory 
parts  opening  into  one  duct ; at  their  bases  is  delicate  connective 
tissue,  adenoid  tissue,  and  blood-vessels.  Below  the  closed  ends 
of  the  glands,  in  the  cat,  a clear  homogeneous  layer  of  condensed 


268  PRACTICAL  HISTOLOGY.  [XXIV. 

connective  tissue,  and  under  this  two  or  three  thin  layers  of  non- 
striped  muscle — the  muscularis  mucosae.  The  clear  layer  of  con- 
densed tissue  is  not  present  in  the  rabbit,  dog,  or  man. 

(6.)  The  submucous  coat,  composed  of  loose  connective  tissue, 
with  large  blood-vessels  and  a few  fat-cells.  If  the  mucous 

membrane  be  folded  and  rugae  are 
present,  the  connective  tissue  will 
be  seen  to  run  up  into  the  folds. 

(c,)  The  muscular  coat  consists 
of  two  or  three  layers  of  smooth 
muscular  fibres.  The  appearance 
varies  according  to  the  manner  in 
which  the  plane  of  the  section 
cuts  them,  for  the  stomach  has 
an  outer  longitudinal  and  an  inner 
circular,  and  in  some  places  an 
oblique  muscular  coat. 

(d.)  Outside  all  a thin  layer — 
peritoneum  or  serous  coat — con- 
sisting of  fibrous  tissue  covered  by 
a layer  of  endothelium. 

(e.)  (H)  Study  specially  the 
mucous  coat.  Observe  the 
columnar  epithelium  lining  the 
stomach,  and  dipping  into  the 
mouths  and  lining  the  ducts, 
which  are  slightly  funnel-shaped. 
The  cells  are  tall  and  narrow,  and 
if  the  section  be  not  too  thin,  the 
ends  of  them  may  be  seen  as 
very  small,  clear,  sharply-defined, 
polygonal  areas.  As  they  secrete 
mucus,  they  have  been  called  mucous  cells.  The  upper  two-thirds 
or  so  of  the  cell  is  much  clearer  than  the  lowest  third,  which  tapers 
somewhat,  and  is  more  granular.  Each  cell  contains  an  oval  nucleus 
in  its  lowest  third,  near  its  attached  end. 

(/.)  Select  a fundus  gland  (fig.  257).  Trace  its  duct  down- 
wards, and  note  that  perhaps  it  is  comnion  to  two  secretory 
portions.  In  the  true  secretory  part,  note  the  large  ovoid,  border, 
parietal,  or  outer  cells,  lying  next  the  basement  membrane, — ovoid 
in  shape,  granular  in  appearance,  and  containing  an  ovoid  nucleus. 
They  do  not  form  a continuous  layer,  but  bulge  out  the  basement 
membrane  here  and  there.  At  the  upper  part  of  the  gland-tube  the 
parietal  cells  are  smaller,  and  placed  nearer  each  other,  while 
towards  the  fundus  or  base  of  the  gland  they  are  larger  and  further 


Fig.  256. — V.S.  Wall  of  Human  Stomach. 
I!.  Epithelium ; G.  Glands ; Mm,  Mus- 
cularis  mucos00,  x 15. 


XXIV.] 


THE  STOMACH. 


269 


apart,  the  interval  between  any  two  being  occupied  by  the  inner  ot 
central  cells.  The  parietal  cells  are  deeply  stained  by  aniline-blue 
and  carmine,  and  osmic  acid,  while  in  a logwood-stained  section  the 
inner  cells  are  usually  more  deeply  stained. 

Observe  a continuous  layer  of  cells — inner,  central,  or  chief — 
lying  internal  to  the  parietal  cells.  They  are  smaller,  and  belong 
more  to  the  columnar  type  of  cell,  but  they  are  not  of  uniform  height 
throughout ; thus  they  are  shorter  over  a parietal  cell,  and  larger 
between  two  parietal  cells.  Each  cell  contains  a spherical  nucleus. 

The  lumen  of  the  gland-tube  is  very 
narrow.  The  secretory  part  is  much  longer 
than  the  duct. 

(g.)  Arrange  the  preparation  so  that  the 
lower  ends  of  the  glands  come  into  view. 

Note  their  closed  extremities,  and  if  the 
granular  epithelium  have  shrunk  somewhat, 
the  basement  membrane  of  each  gland-tube 
may  be  seen  ; very  probably  transverse  or 
oblique  sections  of  the  lower  part  of  the 
gland  will  be  found  (fig.  257). 

(/l)  At  the  bases  of  the  gland-tubes,  but 
outside  their  basement  membrane,  note  the 
delicate  connective  tissue  or  adenoid  tissue, 
with  a number  of  leucocytes.  If  the  section 
be  from  a cat’s  stomach,  a clear  homogene- 
ous layer  runs  outside  this. 

(^.)  Outside  this  the  muscularis  mucosae 
composed  of  at  least  two  layers  of  non-striped 
muscle  arranged  in  opposite  directions. 

Especially  in  a balsam  specimen,  fine  strands 
of  muscular  fibres  are  always  to  be  seen 
passing  from  the  muscularis  mucosae  in- 
wards towards  and  between  the  glands. 

Usually  these  fine  strands  pass  inwards 
between  groups  of  glands.  In  the  osmic 
acid  preparations,  the  parietal  cells  are  much 
darker  than  the  central  cells. 

(./.)  There  is  nothing  particular  to  note  in  the  structure  of  the 
other  coats. 

2.  T.S.  Fundus  Glands. — Make  a section  parallel  to  the  surface 
of  the  mucous  membrane,  stain  with  logwood,  or  picro-carmine  and 
aldehyde-green,  or  use  an  osmic  acid  preparation. 

(H)  Observe  that  all  the  gland-tubes  are  not  cut  at  the  same 
level ; some  are  divided  through  the  duct,  others  through  the 
secretory  part  of  the  tube.  In  the  latter  observe  the  large  parietal 


Em.  257. — V.S.  Mucous  Mem- 
brane of  the  Stomach,  Cat. 
Osmic  acid. 


270 


PRACTICAL  HISTOLOGY. 


[xxiv. 


cells — few  in  number — and  inside  these  a complete  layer  of  inner 
cells  bounding  the  small  lumen  of  the  tube  (fig.  258).  The  glands 

are  arranged  in  groups  as  shown 
by  the  connective  tissue  sur- . 
rounding  several  tubes.  This 
is  very  marked  in  the  pig. 

3.  Fresh  Fundus  Glands. — 
From  the 
mucous 
membrane 
of  the  sto- 
mach of  a 
newly- 
k i 1 led 
guinea-pig 
make  a 
thin  verti- 
cal section 
with  scis- 
sors, and  tease  it  in  normal  saline  to  isolate 
some  of  the  glands.  This  animal  is  selected 
because  its  gland-tubes  are  short,  but  a rabbit 
does  very  well.  The  elevations  of  the  base- 
ment membrane  due  to  the  bulging  of  the 
parietal  cells  are  usually  well  seen. 

4.  V.S.  Pyloric  Mucous  Membrane  (fig. 

259). — {a,)  (L)  Observe  the  same  arrangement 
of  coats  as  in  the  cardiac  end ; but  the  muscu- 
lar coats  are  thicker  ; the  mouths  of  the  gland- 
tubes  are  wider  and  longer,  the  secretory  part 
more  branched  and  sliorter  than  in  the  cardiac 
portion.  There  is  also  much  more  connective 
tissue  between  the  glands. 

{h.)  (H)  The  wide  mouth  of  the  glands,  lined 
by  narrow*  columnar  epithelium,  the  secretory 
part  consists  of  several  tubes  opening  into  one 
gland-duct.  The  secretory  part  lined  by  a 
single  layer  of  cells,  somewhat  cubical,  but 

there  are  no  parietal  cells.  259.  — v.s.  Pyioric 

/ \ ^ j • j • Mucous  Membrane.  D. 

(c.)  There  is  much  more  connective  tissue  Duct ; Secretory  part 

between  the  tubes.  A mass  of  adenoid  tissue  t4si|e^^  *’ 

— solitary  follicle — may  be  seen  in  the  deeper 

part  of  the  mucous  coat.  In  some  cases  its  pointed  apex  may  be 

seen  reaching  nearly  to  the  surface  of  the  mucous  membrane. 

5.  Osmic  Acid  Preparations. — Few  reagents  are  so  good  for 


Fig.  258. — T.S.  Duct  of  Gland  of  Fundus,  a. 
Chief,  h.  Parietal  cells;  r.  Adenoid  tissue; 
c.  Capillaries. 


XXIV.] 


THE  STOMACH. 


271 


fixing  the  cells  of  the  glands  of  the  mucous  membrane  (mount  in 
Farrant’s  solution).  In  sections  of  the  fundus,  the  outer  cells  are 
more  deeply  stained,  and  so  are  readily  distinguished.  The  columnar 
cells  lining  the  ducts  of  the  cardiac  and  pyloric  glands  may  be 
blackened  by  the  osmic  acid  where  they  contain  mucigen. 

6.  Blood-Vessels  of  the  Stomach. — Make  Y.S.  of  an  injected 
stomach  embedded  in  paraffin.  Mount  in  balsam.  They  must  not 
be  too  thin.  iNote  the  large  vessels  in  the  submucous  coat,  and 
from  them  smaller  vessels  proceeding  vertically  upwards,  splitting 
up  into  capillaries  between  the  tubules,  and  forming  a capillary 
network  round  the  mouths  of  the  glands.  Beautiful  plates  by  Mall.^ 

7.  Pyloro-Duodenal  Mucous  Membrane. — It  is  well  to  have  a 
section  through  the  pyloric  valve  to  include  the  mucous  membrane 
on  its  gastric  and  duodenal  boundaries.  To  ensure  this,  the  mucous 
membrane  or  entire  thickness  of  the  stomach  must  be  pinned  out 
on  cork  before  it  is  hardened  by  any  of  the  methods,  e.r/.,  sublimate 
(p.  267).  It  is  treated  like  the  sections  of  the  stomach,  and  does 
best  when  stained  with  eosin-hsematoxylin.  On  one  side  of  the 
thickened  pyloric  valve — the  increased  thickness  being  due  chiefly 
to  an  increase  of  the  circular  muscular  fibres — one  sees  the  pyloric 
structure,  and  on  the  other  that  of  the  duodenum.  The  tubular 
glands  of  the  stomach  are  confined  to  the  mucous  membrane,  but 
the  acini  of  Brunner’s  glands  lie  in  the  submucous  coat  of  the 
duodenum. 

8.  Junction  of  (Esophagus  and  Stomach.— Similar  preparations 
may  be  made.  The  transition  from  the  oesophageal  mucous  mem- 
brane with  stratified  epithelium  and  few  glands  in  the  oesophagus 
to  that  of  the  stomach  with  its  columnar  epithelium  and  mucous 
glands  is  sudden  and  abrupt. 


ADDITIONAL  EXERCISES. 

9.  Double-Staining  the  Stomach. — Harden  the  stomach  in  Muller’s  fluid, 
and  stain  (24  hours)  the  sections  in  iiidigo-carmine  (p.  67) ; afterwards  extract 
the  excess  of  pigment  by  steeping  them  for  half  an  hour  in  a saturated  solution 
of  oxalic  acid.  Mount  in  Farrant’s  solution  or  balsam.  The  parietal  cells  are 
grey  or  blue,  the  central  ones  coloured,  but  with  red  nuclei,  and  the  smooth 
muscle  blue  with  red  nuclei. 

10.  Ehrlich  Biondi  Fluid. — Stain  in  this  fluid  sections  of  the  mucous  mem^ 
brane  fixed  in  sublimate  (saturated  in  .6  per  cent.  NaCl).  If  the  fluid  be  kept 
for  some  time,  an  additional  quantity  of  acid-fuchsin  must  be  added  to  it. 
The  parietal  cells  are  red  and  their  nuclei  blue  ; tbe  chief  cells  are  scarcely- 
stained  at  all,  but  their  nuclei  are  faintly  blue.  Vacuoles  may  be  seen  in  some 
of  the  outer  cells. 

11.  Aniline-blue  and  Safranin. — Sections  of  the  cardiac  end  fixed  in 

^ Vessels  and  Walls  of  Dog’s  Stomach,  Johns  Hopkins  Hosp.  Rep. , vol.  i.  1893. 


PRACTICAL  HISTOLOGY. 


272 


[xxv. 


mercuric  chloride  are  stained  with  aniline-blue  and  then  with  gafranin.  In 
balsam  preparations  the  parietal  cells  are  pale  blue,  the  inner  cells  red. 

12.  Isolated  Cells  of  Gastric  Glands. — Macerate  fragments  of  the  gastric 
mucous  membrane  of  a newt  in  5 per  cent,  ammonium  chromate  (24-48  hours). 
Stain  in  picro-carmine,  and  tease  in  glycerine.  Numerous  isolated  cells  from 
the  ducts  and  secretory  parts  of  the  glands  are  obtained. 


LESSON”  XXV. 

THE  SMALL  AND  THE  LARGE  INTESTINE. 
SMALL  INTESTINE. 

It  has  four  coats — mucous,  submucous,  muscular,  and  serous. 

Study  specially  the  mucous  coat.  In  man,  in  certain  parts,  there 
are  permanent  folds  of  the  mucous  membrane — valvulse  con- 
niventes — and  everywhere  the  surface  is  beset  with  small  conical 
elevations — villi.  At  the  bases  of  the  villi  is  a layer  of  simple 
tubular  glands — Lieberkiihn’s  glands— embedded  in  an  adenoid 
tissue  matrix.  Underneath  this  is  the  muscularis  mucosae,  usually 
consisting  of  three  thin  layers  of  smooth  muscle. 

A villus  (.  5-3  mm.  long)  consists  of  a central  core,  enclosing  a 
lacteal,  and  covered  by  a single  layer  of  columnar  epithelium  with 
goblet-cells  (Lesson  V.).  The  body  of  a villus  consists  of  a tissue 
like  adenoid  tissue  with  leucocytes  and  other  cells.  The  central 
lacteal  is  really  a lymphatic,  and  begins  by  a closed  extremity. 
Several  strands  of  smooth  muscle  pass  from  the  muscularis  mucosae 
into  the  villus,  and  reach  its  upper  extremity.  It  is  very  vascular, 
and  the  blood-vessels  are  distributed  immediately  under  the  epi- 
thelium. 

The  mucous  membrane  also  contains  solitary  follicles,  and  in 
some  situations  Peyer’s  patches,  the  latter  are  most  abundant  in 
the  ileum. 

Methods. — Make  transverse  sections  of  the  small  intestine  of  a 
cat  or  dog  hardened  in  a mixture  of  potassic  bichromate  and 
chromic  acid ; Klein’s  fluid ; per  cent,  chromic  acid ; Kleinen- 
berg’s  fluid  ; or  mercuric  chloride. 

(i.)  Stain  a section  in  hsematoxylin  and  mount  it  in  balsam,  or 
stain  another  in  picro-carmine  and  mount  it  in  Earrant’s  solution. 

(ii.)  All  the  parts  and  their  relations  are  best  preserved  by 
staining  in  bulk  in  borax-carmine  and  cutting  in  paraffin,  or  embed 
in  paTaffin,  cut,  fix  on  a slide,  and  then  stain. 


XXV.] 


SMALL  INTESTINE. 


273 


(iii.)  Cut  sections  by  freezing  and  place  some  in  i per  cent,  osmic 
acid  (24  hours).  This  sharpens  the  outlines  of  many  of  the  structures. 

(iv.)  It  is  convenient  in  teaching  to  give  a . complete  transverse 
section  of  the  small  intestine  of  a small  animal,  e.g.,  mouse  or 
kitten.  In  herbivora  the  wall  of  the  gut  is  very  thin.  Stain  in 
bulk  and  cut  in  paraffin.  Flemming's  fluid  is  an  excellent. “ fixing" 
reagent  both  for  the  small  and  large  intestine.  Stain  the  sections 
in  safranin. 

1.  T.S.  Small  Intestine  (L). — Observe  the  serous,  muscular, 
submucous,  and  mucous  coats  (fig.  260). 

{a.)  In  the  mucous  coat,  the  surface  beset  with  small  conical 
projections — villi — which,  if  they  are  contracted,  exhibit  wrinkles 
on  their  surface.  At  the  bases  of  the  villi  a single  layer  of  simple 
test-tube-like  glands — glands  of  Lieberkiilm — or  intestinal  glands, 
embedded  in  an  adenoid  tissue  matrix.  Outside  this  the  muscu- 
laris  mucosae. 

{h.)  The  submucous  coat,  composed  of  fibrous  tissue  with  nerves 
and  blood-vessels. 

(c.)  The  muscular  coat,  composed  of  two  layers,  an  outer  longi- 
tudinal and  an  inner  circular  layer.  In  the  cat,  the  latter  is  much 
thicker  than  the  former. 

{d.)  The  serous  coat. 

(e.)  (H)  Study  a villus.  Observe  the  single  layer  of  granular 
nucleated  columnar  epithelium  covering  it,  each  cell  with  its  free 
end  covered  by  a clear  disc,  with  vertical  striae  (fig.  260).  The 
succession  of  these  free  clear  borders  looks  like  a clear  hem  round 
the  circumference  of  the  villus.  Occasionally  leucocytes  may  be 
seen  between  the  cells. 

if.)  The  goblet  cells,  chalice,  or  caliciform  cells  scattered  among 
the  former  (fig.  260).  They  may  be  seen  from  the  side,  or  their 
open  rounded  mouths  may  be  directed  toward  the  observer.  When 
seen  from  the  side,  they  are  ovoid,  with  a larger  and  clearer  upper 
part  containing  mucigen,  and  a smaller,  lower,  granular,  nucleated 
part  (Lesson  Y.).  Sometimes  a plug  of  mucus  may  be  seen  protruding 
from  the  mouth  of  a cell.  It  is  stained  blue  with  logwood  and  brown 
with  Bismarck  brown.  If  the  mucous  glands  be  active  and  the  fresh 
tissue  be  fixed  in  osmic  acid,  then  the  plug  of  mucus  is  black. 

(^.)  In  the  centre  of  the  villus  a vertical  space,  the  radicle  of  a 
lacteal,  with  a thin  nucleated  wall.  The  substance  of  the  villus 
consists  of  adenoid  tissue  beset  Avith  leucocytes.  Close  under  the 
epithelium,  perhaps,  sections  of  capillaries,  and  a little  farther  in 
one  or  more  strands  of  non-striped  muscle,  which  can  be  traced  to 
the  apex  of  the  villus,  and  downwards  to  the  muscularis  mucosae 
(fig.  260,  MM). 

(A)  The  glands  of  Lieberkuhn  lined  by  short  columnar  nucleated 
25  S ' 


274 


PRACTICAL  HISTOLOGY. 


[xxv. 


cells,  the  nucleus  near  the  attached  end  of  the  cell.  There  is 
a gradual  transition  from  these  cells  to  those  covering  the  villi. 
A clear  border  may  be  seen  on  their  free  ends.  Bizzozero  has 
called  these  “ protoplasm  cells  ” to  dis- 
tinguish them  from  the  goblet-cells  which 
lie  amongst  them.  Goblet-cells,  however, 
are  far  more  abundant  in  the  large  intestine. 
The  lumen  of  each  gland  is  distinct,  and, 
especially  if  the  lining  cells  be  raised 
slightly,  the  basement  membrane  of  the 
gland-tube  may  be  seen.  Between  the 
gland-tubes  numerous  leucocytes  and 
adenoid  tissue ; in  fact,  the  glands  are  set 
in  a mesh  work  of  this  tissue. 

(i.)  The  nmscularis  mucosae  sends  deli- 
cate processes  into  the  villi.  This  is  best 
recognised  in  balsam  specimens,  by  the 
arrangement  of  the  fusiform  nuclei  of  the 
smooth  muscle  cells. 

(j.)  A solitary  gland  or  a Peyer^s  patch 
may  be  cut,  but  it  is  better  to  have  special 
preparations  for  these. 

2.  Peyer’s  Patches  or  Agminated 
Lymph  Follicles. — (i.)  Make  V.S.  through 
a hardened  Peyer’s  patch  (sublimate  or 
alcohol).  Stain  with  eosin  and  haemato- 
xylin  and  mount  in  balsam. 

(ii.)  Fix  a Peyer’s  patch  of  a rabbit  or 
guinea-pig  in  Flemming’s  fluid.  Fix  a sec- 
tion on  a slide  and  stain  it  first  in  i per  cent, 
aniline-blue  (watery).  Wash  out  in  i per 
cent,  ammonia,  then  in  .5  per  cent.  HCl, 
and  stain  in  safranin  (Garbini).  Mount 
in  balsam. 

(L)  Observe  a group  of  oval  or  roundish 
masses  of  adenoid  tissue  crowded  with 
leucocytes  confined  to  one  side  of  the  gut. 
The  conical  apices  of  some  of  them  may  be 
seen  projecting  upwards  quite  to  the  mucous  surface,  covered  only 
by  a layer  of  columnar  epithelium.  Between  the  epithelial  cells 
may  be  seen  colourless  corpuscles  which  have  wandered  from  the 
adenoid  mass.  The  lower  ends  of  the  masses  usually  pass  down 
into  the  submucous  coat.  'No  villi  exist  over  the  apices  of  these 
masses  of  adenoid  tissue  if  they  project  well  into  the  mucous  layer. 
If,  however,  they  do  not,  but  exist  merely  as  rounded  masses  of 


Fig.  260. — T.S.  Small  Intestine 
(Cat),  r.  Villi;  LG.  Lieber- 
kiihn’s  glands;  MM.  Mus- 
cularis  mucosae ; C and  L. 
Circular  and  longitudinal 
fibres  of  muscular  coat;  S. 
Serous  coat. 


XXV.] 


SMALL  INTESTINE. 


275 


adenoid  tissue  in  the  submucous  coat,  then  they  present  the  appear- 
ance seen  in  fig.  261. 

A solitary  follicle — exactly  like  one  of  the  numerous  follicles 
which  compose  a Peyer's  patch — may  be  seen  (fig.  262). 

3.  Blood  Vessels  of 
the  Small  Intestine. — 

Make  a pretty  thick 
transverse  section  of  a 
well-injected  small  in- 
testine (cat).  Cut  in 
paraffin  and  mount  in 
balsam. 

(L)  The  mucous  coat 
is  the  most  vascular 
part ; the  larger  vessels 
lie  in  the  submucous 
coat,  and  few  vessels  in 
the  muscular  coat. 

(H)  An  artery  runs 
to  the  upper  part  of 
each  villus  and  gives 
off  a plexus  of  capil- 
laries ; a vein  on  the 
opposite  side.  A rich 
plexus  of  capillaries 
between  Lieberkiihn’s 
glands.  If  the  section 
pass  through  a solitary 
follicle,  note  that  the 
capillaries  pass  into  it 
and  form  loops  (fig. 

262). 

The  general  distribu- 
tion of  the  blood-vessels 
is  shown  in  fig.  263, 
which  shows  how  the 
blood  reaches  the  vari- 
ous coats. 

4.  Surface  View  of 
Injected  ViUi. — To  see  the  general  arrangement  of  the  blood-vessels 
in  the  mucous  membrane,  inject  the  blood-vessels  of  a rabbit  with 
a red  gelatine  mass.  Mount  in  balsam  a part  of  the  wall  of  the 
small  intestine,  placing  the  mucous  surface  uppermost. 

(L)  Note  the  villi,  and  trace  the  artery  to  its  origin  from  a larger 
artery  in  the  submucous  coat  (fig.  264)  The  artery  runs  on  one 


Fia.  261. — Longitudinal  Section  through  a Peyer’s  Patch 
of  the  Small  Intestine  of  a Dog. 


276 


PRACTICAL  HISTOLOGY. 


[xxv. 


side  of  the  villus  quite  to  the  apex  of  the  latter,  and  the  vein — a 
wider  vessel — descends  on  the  opposite  side.  There  is  a dense 
plexus  of  capillaries  (C)  placed  close  under  the  epithelial  covering. 
The  best  figures  of  the  blood-vessels  of  the  intestine  are  to  be  found 
in  MalTs  paper.^ 

5.  Nerve-Plexuses  in  Intestine.— (a.)  Gold  Chloride  Method.— 
Wash  the  small  intestine  of  a rabbit  with  normal  saline,  fill  it  with 

Villi  with  Solitary 

blood-vessels.  follicle. 


Mucous  mem- 
brane with 
villi 


Blood-vessels, 


Muscular  coat. 


PlGl.  262. — Mucous  Membrane  of  Small  Intestine  (Rabbit),  Injected,  showing 
villi  and  a solitary  follicle,  x 50. 

lemon-juice  or  5 per  cent,  arsenic  acid,  and  leave  it  in  lemon-juice 
or  acid  for  five  minutes.  Allow  the  juice  to  escape,  wash  the  gut 
in  water,  fill  it  with  i per  cent,  gold  chloride,  and  place  it  in  per 
cent,  gold  chloride  solution  (30  mins.).  Wash  it  in  water,  and 
keep  it  in  the  dark  in  25  per  cent,  formic  acid  (48  hours).  It  is 
now  easy  to  separate  the  coats  of  the  intestine  from  each  other. 
Wash  in  water  to  remove  all  the  acid,  and  with  a pair  of  forceps 
strip  off  the  longitudinal  muscular  coat  (Auerbach^s  plexus  adheres 
to  this) ; it  separates  quite  easily  from  the  circular  coat.  Preserve 
what  remains.  Mount  a small  part  of  the  longitudinal  muscular 
coat  in  Farrant’s  solution. 

{h.)  Methylene-hlue  Method  (p.  284). 

^ “ Die  Bhit-  u.  Lj^mpli-wege  im  Diinndarm  des  Hundes,”  Ahhand.  d.  math,- 
phys.  Classed,  K.  Sachs.  Gesell,  d.  Wissens.^  Bd.  xxiv.,  1887. 


XXV.] 


SMALL  INTESTINE. 


277 


Glands. 


6.  Auerbach’s  Plexus  lies  between  the  muscular  coats,  but  when 
they  are  separated  it  usually  adheres 
to  the  longitudinal  coat.  The  general 
arrangement  of  the  plexus  can  be 
seen  with  the  naked  eye  (fig.  265). 

(L)  The  polygonal  meshwork  of 
purplish  stained  fibres,  with  slight 
swellings  at  the  points  of  intersection 
(fig.  266). 

(H)  The  meshes  are  so  large  that 
only  a part  of  them  comes  into  the 
field  of  view  at  once.  !N^ote  the  non- 
medullated  nerve-fibres,  and  at  the 
nodes,  groups  of  nerve-cells.  From 
the  plexus  numerous  fibres  are  given 
off  to  supply  the  smooth  muscle  of 
the  intestine. 

7.  Ganglionic  Cells. — In  a vertical 
section  of  the  gut  (prepared  as  in  1) 
look  for  groups  of  ganglionic  cells  in 
the  submucous  coat  and  others  be- 
tween the  two  layers  of  muscular 
fibres  (fig.  267). 

8.  Meissner’s  Plexus.  — Spread 
what  remains  after  removal  of  the 
longitudinal  muscular  coat  on  a slip  of  glass,  mucous  surface 


Fig.  263. — Scheme  of  the  Distribution 
of  Blood-Vessels  in  the  Small  In- 
testine of  a Dog.  M.M,  Muscularis 
mucosae. 


Fig.  264.— Injected  Villi  of  Small  Intestine  of  Rabbit,  seen  from  above  and  laterally. 

A.  Artery ; V.  Vein  ; C.  Capillary  network. 

uppermost.  With  a knife  gradually  scrape  away  the  mucous  coat. 


2/8 


PRACTICAL  HISTOLOGY. 


[xxv. 


With  care — observing  the  preparation  from  time  to  time  under  a 
low  power  of  the  microscope — the  progress  of  the  process  of  denu- 
dation can  he  easily  observed.  By  and 
by  the  plexiform  arrangement  of  Meissner’s 
submucous  plexus  will  be  seen.  Mount 
in  Farrant’s  solution  (tig.  268). 

(L)  Observe  the  large  wide  meshes  of 
the  plexus.  Compare  the  general  arrange- 
ment of  the  plexus  with  that  of  Auer- 
bach’s plexus.  The  fibres  are  finer,  and 
the  groups  of  nerve-cells  smaller. 

(H)  Note  the  ganglionic  nerve-cells  at 
the  nodes  (fig.  268). 

9.  Brunner’s  Glands. — These  glands 
are  confined  to  the  duodenum.  Proceed 
exactly  as  recommended  for  the  small  in- 
testine. Stain  a section  with  logwood  and 
use  eosin  as  a counter  stain.  This  gives 

excellent  results. 

Perhaps  the  best  method  is  to  slit  up  the  duodenum  longitudi- 


FlG.  265.  — Auerbach’s  Plexus 
(Rabbit).  Lemon-juice  and 
gold  chloride,  x 8. 


Fig.  266.— Auerbach’s  Plexus  (Dog). 


nally,  pin  it  out  on  a cork  plate,  and  fix  it  in  mercuric  chloride  (an 
hour  at  40°  C.  in  a thermostat,  or  24  hours  at  ordinary  tempera- 


XXV.] 


SMALL  INTESTINE. 


279 


ture).  The  surplus  HgCl2  is  extracted  with  water  at  30°  and 
afterwards  with  alcohol  at  40**  C.  It  is  then  removed  from  the 
cork  plate  and  hardened  in  alcohol.  Embed  and  cut  in  paraffin. 
Kuczyhski^  has  investigated  these  glands  in  a great  number  of 


Fig.  267.— Auerbach’s  Plexus  in  Section,  a.  Ganglionic  cells  ; h.  Nerve-fibres;  c.  Circular, 
and  d.  Longitudinal  muscular  fibres. 

animals  and  man,  and  tried  a great  variety  of  stains.  Kleinenberg’s 
fluid  is  also  very  good  as  a fixing  reagent. 

(L  and  H)  Note  villi,  Lieberkiihffis  glands  and  the  usual  intes- 
tinal coats.  In  the  submucous  coat,  which  is  thick,  are  the  acini 


Fig.  268.— Meissner’s  Plexus  Intestine  (Rabbit).  Lemon-Juice  and  gold  chloride. 

of  Brunner’s  glands  (fig.  269),  each  composed  of  a basement  mem- 
brane lined  by  cubical  or  short  columnar  cells.  A duct  from  each 


^ InternaL  Monats,  f,  Anat.  u,  Fhys.^  vii.,  1890,  419. 


28o 


PRACTICAL  HISTOLOGY. 


[xxv. 


gland  perforates  tlie  muscularis  mucosse,  passes  np  between  the 

intestinal  glands,  and  opens 
on  the  free  surface  at  the 
bases  of  the  villi.  It  has  a 
distinct  lumen,  and  is  lined 
by  low  cubical  epithelium. 

10.  Fresh  Villus. — Pre- 
ferably that  of  a mouse. 
Examine  in  normal  saline. 


LAEGE  INTESTINE. 

Methods. — Prepare  it  in 
the  same  way  as  the  smkll 
intestine,  and  make  vertical 
transverse  sections.  The 
details  of  the  structure  will 
necessarily  vary  with  the 
animal  used,  but  perhaps  the 
cat  is  as  convenient  an 
animal  as  any  to  employ. 
I can  strongly  recommend 
Flemming’s  fluid.  Sections 
to  be  stained  with  safranin. 

11.  T.S.  Large  Intestine  (fig.  270). — As  the  large  intestine  is 
wide,  it  is  necessary  in  making  the  section  to  select  a portion  of  the 
great  gut  which  shows  the  longitudinal  coat.  In  man,  the  coat 
is  not  a continuous  one,  the  longitudinal  fibres  being  grouped  for 
the  most  part  into  three  flat  bands  of  fibres. 

(L)  Beginning  from  without  inwards,  observe  four  coats.  In 
the  muscular  coat,  note  the  peculiarities  of  the  longitudinal  coat 
and  the  thick  well-marked  continuous  circular  coat.  There  is 
nothing  special  about  the  submucous  coat,  unless  it  be  the  exist- 
ence of  solitary  follicles,  which,  however,  are  not  confined  to 
the  large  intestine.  The  mucous  coat  is  characterised  by  negative 
characters.  There  are  no  villi.  It  may  exhibit  folds,  into  each 
of  which  there  runs  a projection  of  the  submucous  coat. 
Lieberkiihn’s  glands,  cut  vertically,  and  some  of  them  obliquely  or 
horizontally. 

(H)  In  the  mucous  coat  the  glands  of  Lieberkiihn,  larger  than 
those  of  the  small  intestine,  and  lined  by  cells — protoplasm  cells — 
with  nuclei  near  their  attached  ends.  Amongst  them  are  very 
many  goblet-cells  (fig.  271). 


Fig.  269.— V.S.  Duodenum  of  Cat. 

and  longitudinal  layers  of  muscle;  Lg.  Lieber- 
kiihn’s  glands;  Bg,  Brunner’s  glands;  g. 
Ganglion-cells ; v.  Villi. 


xxv.j 


LARGE  INTESTINE. 


28  J 


Epithelium. 


Mucous 

membrane. 


Capillary. 


Solitary 

follicle. 


Circular 

fibres. 

Muscular 

coat. 


Longitudi- 
nal fibres. 


Eia.  270. — L.S.  Large  Intestine. 

12.  Vermiform  Appendix  (Rabbit). — This  is 
instructive  on  account  of  the  lymphatic  follicles 
it  contains  and  the  micro-organisms  contained 
in  these  follicles  (p.  284).  Fix  it  in  mercuric 
chloride  and  treat  it  as  recommended  for  the 
other  parts  of  the  intestine.  The  lymph-sinuses 
are  readily  injected  by  the  “puncture’’  method 
with  watery  solution  of  Berlin-blue.  Fix  in 

Muller’s  fluid  and  harden  in  alcohol. 

The  lymph  follicles  are  arranged  in  two  or 
three  rows,  the  one  inside  the  other,  but  the 
innermost  layer  does  not  project  beyond  the  level 
of  the  mucous  membrane.  The  apices  of  the 
inner  row  are  covered  only  by  cylindrical  epi- 
thelium. 


ElG.  271. — Lower 
End  of  a Lieber- 
kiihn’s  Gland, 
Large  Intestine 
of  Dog.  G,  Gob- 
let-cells. 


282 


PRACTICAL  HISTOLOGY. 


[xxv 


ADDITIONAL  EXERCISES. 


13.  T.S.  Villus. — Stain  a hardened  mucous  membrane  (with  villi)  of  a dog 
or  cat  ‘‘in  bulk”  in  borax-carmine  or  Kleinenberg’s  logwood,  embed  and  cut 
T.S.  in  paraffin,  fix  on  a slide,  remove  the  paraffin  with  turpentine,  clarify 
with  clove-oil,  and  mount  in  balsam.  Many  of  the  villi  will  be  cut  obliquely. 

(H)  Observe  the  lacteal  (L)  in  the  centre,  and  round  it  the  structure  of  the 
stroma  of  the  villus,  with  several  groups  of  non-striped  muscle-cells  (m)  close 
to  and  surrounding  the  lacteal.  In  some  animals  (dog)  there  is  a double  row 
of  these  smooth  muscles  (fig.  272).  The  capillaries  immediately  under  the 

epithelium  (c),  the  smooth  muscular 
fibres  parallel  to  the  lacteal,  and  the 
stroma — composed  of  anastomosing 
fine  trabeculae  with  parenchymatous 
cells  and  leucocytes — make  up  the 
elements  present  in  the  core  of  a 
villus. 

Great  differences  exist  in  the  rela- 
tive size  of  tlie  stroma  and  lacteal  in 
villi.  In  the  dog  and  cat  the  lacteal 
is  relatively  small,  and  the  stroma 
abundant ; in  the  rabbit  the  lacteal 
is  very  large,  and  the  stroma  scanty. 

14.  Non-Striped  Muscle  in  Villi.  — 
The  method  of  Kultschitzky  enables 
the  course  of  the  fibres  to  be  more 
clearly  traced.  Harden  (for  twenty- 
four  hours)  a piece  of  dog’s  small  in- 
testine in  the  following  fluid  : — A 
saturated  solution  of  potassic  bichro- 
mate and  copper  sulphate  in  50  per 
cent,  alcohol  (in  the  dark),  to  which,  immediately  before  using,  is  added 
5-6  drops  (to  100  cc.)  of  acetic  acid.  The  preparation  and  fluid  must  be  kept 
in  the  dark.  Complete  the  hardening  in  absolute  alcohol.  Make  sections, 
and  stain  them  in  acid  chloral-hydrate  carmine^  which  is  made  as  follows  : — 
Chloral  hydrate,  10  grams;  hydrochloric  acid  (2  per  cent.),  100  cc.  Add  to 
this  dry  carmine  (.75  to  1.5  gram),  according  to  the  strength  of  stain  desired. 
Boil  for  one  and  a half  hours,  preventing  evaporation  by  means  of  a cooling 
apparatus.  Allow  it  to  cool  and  filter.  If  the  preparations  stained  with  this 
dye  be  washed  in  2 ]>er  cent,  alum,  the  nuclei  and  other  tissues  become  violet. 

Sections  stained  thus  show  the  course  of  the  smooth  muscle  from  the 
muscularis  mucosi®  obliquely  between  Lieberkiihn’s  glands  into  the  villi,  where 
they  are  arranged  in  several  bundles  near  the  lacteal.  They  curve  as  they 
ascend  in  the  villus,  the  concavity  looking  outwards,  and  are  fixed  or  inserted 
close  under  the  e})ithelium.  They  pass  quite  to  the  apex  of  the  villus,  becom- 
ing thinner  as  they  go,  where  they  each  split  up  into  a pencil  of  fibres,  the 
fibres  being  inserted  close  under  the  epithelium. 

15.  Heidenhain’s  Method. — Harden  small  pieces  of  the  small  intestine, 
e.g.,  dog  or  cat  (24  hours),  in  a .5  solution  of  common  salt  saturated  with 
mercuric  chloride.  Place  it  for  twenty-four  hours  in  alcohol  80,  90,  and  95 
per  cent.,  and  finally  in  absolute  alcohol.  Saturate  with  xylol,  embed  in 
paraffin,  and  cut  thin  sections,  which  are  fixed  on  a slide  with  a “fixative.” 
After  the  paraffin  has  been  got  rid  of  by  turpentine  or  xylol,  and  the  turpen- 
tine displaced  by  alcohol,  the  sections  are  stained  on  the  slide  with  Ehrlich- 


FlG.  272.— T.S.  Villus  (Dog).  L.  Lacteal ; 
m.  Muscle ; c.  Capillaries,  x 400. 


XXV.] 


LARGE  INTESTINE. 


283 


Biondi’s  fluid  (p.  81),  diluted  with  40  or  50  volumes  of  water.  It  requires 
10-12  hours  to  stain  the  sections,  which  are  then  mounted  in  bals^im.  This 
preparation  is  particularly  valuable  for  studying  the  various  forms  of  cells  that 
occur  in  the  stroma  of  a villus.^ 

16.  Absorption  of  Fat. — (i.)  Feed  a frog  on  fat  bacon  ; after  two  days  kill 
it,  and  tease  a portion  of  the  mucous  membrane  of  the  intestine  in  normal 
saline,  or  dissociate  it  in  dilute  alcohol.  Observe  the  isolated  columnar  cells 
crowded  with  fine  granules  of  oil,  which  are  blackened  on  the  addition  of 
osmic  acid. 

(ii.)  A better  plan  is  to  stain  the  mucous  membrane  in  osmic  acid  (24  hours), 
and  embed  it  in  paraffin.  It  is  to  be  remembered,  however,  that  steeping  the 


Fig.  273.— Villus  of  Dog’s  In- 
testine, with  Lacteal  and 
Non -Striped  Muscle.  L. 
Lacteal,  x 250. 


Fig.  274. — Section  of  Intestine  of 
Frog.  Absorption  of  fat.  Osmic 
acid. 


mass  or  setdions  of  it  in  paraffin  discharges  in  part  the  black  colour  of  the  fatty 
granules  (p.  33). 

(iii. ) Feed  a rat  on  bread  and  fat  of  bacon  ; kill  it  four  hours  afterwards  by 
means  of  curare.  Harden  the  small  intestine  in  OSO4. 

(H)  Observe  the  projections  like  folds  of  the  mucous  membrane.  The 
columnar  cells  covering  them  are  crowded  with  blackened  particles — fatty 
granules  blackened  by  osmic  acid  (fig.  274). 

17.  Tubular  Glands  of  the  Intestine. — In  studying  these,  different  fixing 
fluids  are  used  according  to  the  animal  selected  (Bizzozero).*^  Amongst  the 
best  fixing  reagents  are  saturated  watery  solution  of  ])icric  acid  or  picro- 
sulphuric  acid  (2  days),  and  wash  in  water  (i  day),  subsequently  hardening  in 
alcohol.  This  is  suitable  for  the  rectum  of  the  mouse  and  dog.  For  the 
duodenum,  either  alcohol  or  Flemming’s  fluid,  or  Hermann’s  fluid  (Lesson 
XXXV.).  They  are  stained  in  safranin  (1-2  hours),  washed  (10-15  secs.)  in 
absolute  alcohol,  and  then  stained  in  haematoxylin.  Cleared  in  bergamot 
oil  and  mounted  in  balsam.  Safranin  stains  the  nuclei  of  the  cells,  but  not 
the  mucin.  The  latter  is  stained  by  the  logwood,  and  one  can  trace  it  from 
the  goblet-cells  passing  into  the  lumen  of  the  tubule.  Mitosis  may  be 
observed  both  in  the  protoplasmic  and  goblet  cells. 

^ Pfluqer's  Archiv,  Supp.  Bd.,  1888. 

2 Archivf.  mik.  Anat.y  p.  325,  1892, 


284 


PEACTICAL  HISTOLOGY. 


[xxv. 


The  mucus  in  tlie  fresh  goblet-cells  occurs  in  the  form  of  granules.  This  is 
best  seen  in  a fresh  preparation  teased  without  the  addition  of  any  fluid,  or  at 
most  in  Muller’s  fluid.  In  sections  fixed  in  Flemming’s  fluid  and  stained  with 
safranin,  the  goblet-cells  are  reddish.  The  network  in  these  cells  may  be 
stained  with  Bismarck-brown. 

Accorling  to  Bizzozero,  the  epithelial  investment  of  the  villi  is  not  renewed 
by  the  proliferation  of  the  cells  covering  it ; but  by  the  proliferation  and 
upward  growth  of  the  cells  lining  Lieberklihn’s  follicles. 

18.  Terminations  of  Nerves  in  Stomach  and  Intestine. — Besides  the  gold- 
chloride  method  (Drasch,^  nerves  of  tlie  duodenum),  and  the  methylene-blue 
methods  as  used  by  Aronstein,-  Golgi’s  silver  method  has  been  recently  applied 
by  Erik  Muller ^ for  this  jairpose.  The  method  used  was  Golgi’s  “rapid 
method”  (Lesson  XXX.),  ^.e.,  osmico-bichromate  solution  and  subsequent 
staining  with  silver  nitrate.  The  nerve- fibres  become  black,  and  numerous 
communications  are  found  to  exist  between  the  plexuses  of  Auerbach  and 
Meissner.  A very  large  number  of  nerve-fibrils  enter  the  villi,  and  are  dis- 
tributed in  them,  reaching  to  the  cylindrical  epithelium  covering  them.  They 
end  free  and  have  not  been  seen  to  end  in  or  between  the  cylindrical  cells. 
Some  end  in  the  smooth  muscular  fibres  of  the  villi.  Other  fibrils  surround 
the  gland  tubes.  It  is  said  that  branched  cells,  like  nerve-cells,  lie  in  the  villi 
{B.  y CayaT). 

19.  Methylene-Blue. — Immediately  after  death  inject  into  the  thoracic 
aorta  of  a guinea-pig  or  rabbit  the  following  fluid; — i gram  methylene-blue 
BX  in  300  cc.  normal  saline.  Open  the  abdomen  and  expose  the  gut  to  the 
air  for  2-3  hours.  Place  a thin  piece  of  the  wall  of  the  small  intestine  in 
picrin-glycerine  of  S.  Mayer  (p.  192)  and  search  for  the  reddish-stained  nerve 
plexus  {S.  Mayer), 

If  a piece  of  the  gut  be  teased  in  picrin-glycerine,  it  is  easy  to  isolate  blood- 
vessels with  their  nerves.  It  is  to  be  noted  that  methylene-blue  not  only 
stains  the  axis-cylinders  of  nerves,  but  also  the  cement  of  epithelial  cells,  so 
that  in  some  respects  it  acts  like  silver  nitrate. 

20.  Vermiform  Appendix  {Rabbit). — Fix  in  Flemming’s  fluid,  Fol’s  solution, 
or  absolute  alcohol. 

A.  For  Mitosis. — (i.)  Stain  the  sections  (5-10  mins.)  in 


(2.)  Wash  in  absolute  alcohol.  (3.)  Immerse  (30-40  secs.)  in  i per  cent,  chromic 
acid.  (4.)  Again  absolute  alcohol  (30-40  secs.) ; and  (5.)  in  chromic  acid. 
Then  in  absolute  alcohol  to  remove  all  surplus  dye.  Balsam.  In  this  way 
the  chromatin  of  the  nuclei  is  stained. 

B.  For  Micro-Organisms. — Stain  as  above  in  (i).  (2.)  Absolute  alcohol 

(5  secs.).  (3.)  Weak  iodine  solution,  ^.e.,  Gram’s  method  (p.  105)  (2  mins.). 
(4.)  Then  alternately  chromic  acid  and  alcohol  as  above.  Balsam  (Bizzozero). 
The  secret  of  getting  good  preparations  is  to  wash  them  well  in  absolute 
alcohol  until  all  surplus  dye  is  removed. 

21.  Peyer’s  Patches  and  Phagocytosis.— (a.)  Harden  a Peyer’s  patch 
(rabbit)  in  absolute  alcohol,  cut  sections  in  paraffin,  and  stain  them  in  alum- 
carmine,  or  in  addition  with  gentian-violet  by  Gram’s  method  (p.  284) 

^ Sitzb.  d.  k.  Akad.  d.  Wissensch.^  Bd.  81,  hi.  Abth.,  Wien,  1880. 

^ Anat.  Anzeiger^  Bd.  ii.,  1887. 

^ Archiv f.  mik.  Anat.^  Bd.  xl.,  1892. 


Gentian-violet  . 
Absolute  alcohol 
Aniline  oil 
Water 


j i i 

80  ,, 


I gram. 
15  cc. 

3 „ 


LIVER. 


285 


XXVI.] 

(Buffer'^).  (H)-  In  the  sections  numerous  small  leucocytes  will  be  found  to 
have  wandered  from  the  lymph  follicles  between  the  epithelial  cells.  There 
are  always  to  be  seen  several  much  larger  cells — mono-nucleated— in  the  lymph 
follicles.  The  smaller  forms — which  may  be  mono-nucleated  or  poly-nucleated 
— have  been  called  microphages,  and  the  largest  macrophages  because  both 
are  capable  of  taking  up  bacteria — dead  and  alive—  into  their  protoplasm  and 
changing  them  by  a process  of  intracellular  digestion.  Transition  forms 
between  microphages  and  macrophages  of  leucocytes  may  be  found.  In  a 
preparation  stained  by  Gram’s  method  the  bacilli — which  aie  found  in  the 
lymph-cells,  but  not  in  the  epithelial-cells — are  stained  deep  blue-violet. 

{h.)  Sections  of  glands  fixed  in  sublimate  may  be  stained  with  Ehrlich- 
Biondi  fluid  and  the  same  kinds  of  lymph-cells  as  occur  in  lymph-glands  are 
found.2  The  most  numerous  are  (i)  small  lymph-cells  with  a nucleus  (stained 
green)  surrounded  by  a small  quantity  of  rose-coloured  protoplasm.  {2.) 
Larger  cells  with  rose-red  protoplasm.  (3.)  Granular  cells  which  seem  to 
correspond  to  eosinophilous  cells.  (4.)  Cells  undergoing  degeneration  ; and 
(5.)  Phagocytes. 


LESSON  XXVI. 

LIVER. 

It  is  composed  of  a large  number  of  lobules  (i  mm.  inch)  in 
diameter),  held  together  by  a greater  or  less  amount  of  connective 
tissue.  Each  lobule  practically  resembles  its  neighbour,  and  is 
composed  of  a mass  of  polyhedral  or  cubical  liver-cells  which  have 
in  relation  with  them  blood-vessels  and  bile-ducts.  The  liver  is 
covered  by  a capsule,  which  sends  processes  into  the  organ  at  the 
portal  fissure,  forming  Grlisson’s  capsule,  which  lies  between  the 
lobules  in  the  portal  canals,  and  surrounds  the  portal  vein,  bile-duct, 
and  hepatic  artery.  The  liver  is  supplied  with  blood  by  the^vena 
portae  and  hepatic  artery ; they  enter  at  the  portal  fissure,  and  the 
former  divides  into  branches  vdiich  ramify  between  the  lobules, 
constituting  the  interlobular  veins.  From  these  veins  capillaries 
pass  into  and  traverse  the  substance  of  the  lobules,  and  converge  to 
a veinlet  in  the  centre  of  each  lobule — the  central  or  intralobular 
veins  or  rootlets  of  the  hepatic  vein,  which  form  sublobular  veins, 
and  these  in  their  turn  form  the  hepatic  vein  which  carries  the 
blood  from  the  liver  to  the  inferior  vena  cava.  The  substance  of 
each  lobule  between  the  capillaries  is  composed  of  liver-cells  (20  /x, 
inch  in  diameter),  which  form  anastomosing  columns,  being 
more  radiate  next  the  centre  of  the  lobule.  Between  the  liver-cells, 

^ Quart.  Jour.  Micr.  Sci.,  xxx.  p.  481. 

2 Hoyer,  Archivf.  mik.  Anat.,  xxxiv.,  1889. 


286 


PRACTICAL  HISTOLOGY. 


[XXVI. 


but  always  separated  from  tlie  blood-capillaries  by  hepatic  cells  or 
part  of  a cell,  is  a fine  polygonal  plexus  of  channels — the  bile 
capillaries,  which  become  continuous  with  interlobular  bile-ducts 
at  the  margin  of  the  lobule.  The  smaller  interlobular  bile-ducts 
unite  to  form  larger  bile-ducts,  which  are  lined  by  columnar  epi- 
thelium, and  in  the  walls  of  the  largest  of  them  are  mucous  glands. 
The  hepatic  artery  supplies  chiefly  the  connective  tissue  between 
the  lobules,  and  accompanies  the  branches  of  the  bile-duct  and 
portal  veins,  so  that  these  three  structures  lie  together  in  portal 
canals. 

Methods. — Harden  portions  of  the  liver  of  a,  e.g.^  pig,  rabbit,  cat, 
and  man  in  Muller’s  fluid  or  2 per  cent,  potassic  bichromate  (10-14 
days).  If  it  be  desired  to  retain  the  chromatin  fibrils  in  the  liver- 
cells  avoid  the  chromic  acid  salts.  Use  sublimate,  and  then 
gradually  increasing  strengths  of  alcohol.  Cut  sections  parallel  to  the 
surface  of  the  organ,  and  others  at  right  angles  to  it,  the  latter  to 
include  the  capsule.  The  sections  can  be  stained  in  hsematoxylin 
and  mounted  in  balsam ; or  picro-carmine,  or  picro-lithium  carmine, 

and  Farrant’s  solution 
may  be'  used.  Staining 
in  bulk  and  cutting  in 
paraffin  is  also  good. 

If  unstained  sections 
are  mounted,  the  out- 
lines of  the  tissues  will 
be  much  better  defined 
if  they  be  soaked  in  i 
per  cent,  osmic  acid  (24 
hours)  previous  to  being 
mounted  in  Farrant’s 
solution. 

1.  Liver  of  Pig 

{Hcematoxylin  and  Bal- 
sam^ or  PicroUtliium 
Carmine^  or  Methylene- 
Blue). 

(a.)  (L)  Observe  the  polygonal  lobules  (fig.  275)  mapped  out  from 
each  other  by  a network  of  septa  of  connective  tissue,  or  Glisson’s 
capsule  (fig.  275,  S).  In  the  centre  of  each  lobule  a small  thin- 
walled  vein,  a rootlet  of  the  intralobular  or  hepatic  vein 
(I.V.). 

(h.)  At  the  ])eriphery  of  each  lobule  sections  of  branches  of  the 
portal  or  interlobular  vein  ; if  j)ossible,  find  a transverse  section  of 
the  latter,  and  it  will  be  found  to  be  accompanied  by  similar  sec- 
tions of  the  bile-duct  (one  or  more)  and  hepatic  artery  (fig.  277, 


Fig.  275.— Liver  of  Pig,  showing  Lobules.  P.C.  Portal 
canal,  containing  bile-duct,  hepatic  artery,  and  por- 
tal vein  (P.  V.) ; S.  Septa ; S.  V.  Sublobular  vein ; I.  V. 
Intralobular  vein. 


XXVI.]  LIVER.  287 

P.Y.).  Branches  of  all  three  structures  always  run  together  in  the 
portal  canals,  and  are  surrounded  by  Glisson’s  capsule. 

(c.)  Capillaries — for  the  most  part  empty — running  from  without 
inwards  in  each  lobule.  Radiating  from  the  hepatic  veinlet  columns 
of  liver-cells,  forming  a network  of  secretory  cells  intertwining  with 
the  capillary  plexus  within  the  lobule. 

{d.)  (H)  The  columns  of  hepatic  cells  radiating  from  the  hepatic 
veinlet,  composed  of  polygonal  or  cubical  cells,  with  granular  con- 
tents and  an  excentrically-placed  spherical  nucleus.  At  the  outer 
part  of  the  lobule  the  network  of  cells  is  more  polygonal,  correspond- 
ing to  the  arrangement  of  the  capillaries.  Between  the  columns  of 
cells,  capillaries,  sometimes  with  a few  blood-corpuscles.  The 
vessels,  however,  are  not  as  distended  as  normal.  Transverse 


Fig.  276. — Columns  of  Liver-Cells 
from  a Starving  Dog. 


branches  running  between  adjoining  capillaries,  and  the  whole 
interwoven  with  the  cellular  network. 

In  a hsematoxylin-stained  section,  note  that  the  nuclei  of  the 
liver-cells  are  stained  to  a less  degree  than  the  other  nuclei.  The 
nuclei  of  the  cells  of  the  connective-tissue,  bile-ducts,  and  capillaries 
are  more  deeply  stained.  In  the  picro-lithium  preparations,  the 
cells  are  yellowish  with  nuclei  red,  but  of  different  shades  of  the 
same. 

With  the  low  power  find  a portal  canal  with  its  contents ; fix 
them  under  the  microscope  and  examine  with  (H). 

(e.)  One  or  more  sections  of  interlobular  bile-ducts,  lined  by  a 
single  layer  of  shorter  or  taller  columnar  cells ; outside  them  con- 
nective tissue,  disposed  circularly  and  continuous  with  that  of 
Glisson^s  capsule  (fig.  277).  A section  of  a branch  of  the  hepatic 
artery  and — the  largest  opening  of  all — of  the  portal  vein.  Some 
slits  may  be  seen  in  Glisson’s  capsule ; they  are  lymphatics. 


288 


PRACTICAL  HISTOLOGY. 


[XXVI. 


2.  Liver  of  Rabbit. — It  is  convenient  to  study  this  liver,  because 
in  some  respects  it  shows  a transition  between  that  of  the  pig  and 
man,  as  it  occupies  an  intermediate  place  with  regard  to  the  demar- 
cation of  its  lobules.  In  it  the  lobules  are  not  nearly  so  well 
defined  by  connective  tissue  as  in  the  pig  or  camel,  while  their 
separation  from  each  other  is  more  definite  than  in  man. 

3.  Human  Liver. — Hsematoxylin,  balsam. 

(L)  Observe  the  greater  fusion  of  the  lobules.  Practically,  the 
arrangements  of  blood-vessels.and  cells  in  other  respects  is  the  same 
as  in  the  rabbit’s  liver  (fig.  278).  If  the  liver  is  anaemic,  the  intra- 


Sublobular 

vein. 


Intralobular 

vein. 


Bile  ducts. 


Interlobular 

veins. 


Fig.  278. — Section  of  Human  Liver,  showing  Liver  Lobules  and  the  Eradiate  Arrange- 
ment of  the  Hepatic  Cells  from  the  Centre  of  each  Lobule,  x 20. 


lobular  blood-capillaries  are  narrow,  and  the  liver-cells  appear  to 
compress  and  narrow  them. 

4.  Liver  of  Frog  or  Newt. — Harden  a small  piece  in  absolute 
alcohol  or  | per  cent,  osmic  acid  (24  hours).  Stain  the  mass  ‘‘in 
hulk”  in  Kleinenherg’s  logwood  or  borax-carmine,  and  mount  in 
balsam.  Mount  the  osmic  acid  sections  in  Tarrant’s  solution  with- 
out staining,  or  stain  them  in  safranin  (48  hours). 

{a.)  (L)  Observe  the  anastomosing  system  of  gland- tubes  made 
up  of  hepatic  cells.  Between  them  the  narrower  blood-capillaries, 
many  of  them  filled  with  blood-corpuscles.  Here  and  there  black 
patches  of  pigment — melanin — especially  in  winter  frogs  (fig.  279). 

{h.)  (H)  Each  cell  is  polygonal,  with  a large  spherical  nucleus ; 
the  contents  may  be  more  or  less  granular,  according  to  the  phase 


LIVER. 


28q 


xxvl] 

of  secretory  activity  of  the  cells.  In  some  conditions  the  granules 
within  the  cells  may  he  arranged  next  the  capillary ; in  others  they 
are  more  regularly  scattered  throughout  the  cell  substance.  Tne- 
cells  are  arranged  round  the  blle-capillaries.  This  is  best  seen  in 
transverse  sections  of  the  latter,  which  appear  as  very  small  circular 
apertures  bounded  by  four  or  five  cells  (tig.  279).  When  the  tubes 
are  cut  longitudinally,  the  bile 
capillaries  are  seen  to  pursue  a 
zigzag  course  between  the  cells, 
but  this  will  be  better  seen  in 
injected  specimens. 

(c.)  The  blood-capillaries  with 
their  nucleated  blood-corpuscles, 
and  note  that  there  is  always  a 
cell  or  part  of  a cell  between 
the  blood-stream  with  its  wide 
lumen,  and  the  bile-channels 
with  very  narrow  lumina.  Any 
fatty  granules  present  in  the 
osmic  acid  section  are  black. 

5.  Blood-Vessels  of  the 
Liver — Opaque  Injections. — Mount  in  balsam  a section  of  a pig’s 
liver,  with  the  P.Y.  injected  with  a red  opaque  mass,  and  the  H.Y. 
with  a similar  yellow  mass.  The  light  from  the  reflector  must  be 
turned  off,  and  light  focussed  on  the  preparation  by  a condenser 
(fig.  16). 

(a.)  (L)  Observe  the  polygonal  lobules,  the  branches  of  the 
portal  veins  around  the  periphery  of  the  lobules,  and  sending  fine 
branches  into  the  latter.  In  the  centre  of  the  lobules  the  hepatic 
veinlets  yellow,  with  capillaries  converging  to  them.  The  capil- 
laries within  the  lobules  partly  filled  with  red  and  partly  with 
yellow  mass,  connecting  the  portal  and  hepatic  venous  systems. 

(b,)  Sometimes  a longitudinal  section  of  a lobule  may  be  seen. 
Trace  the  veinlet  to  a larger  branch  under  the  lobule,  ^.e.,  to  a 
sublobular  vein.  The  sublobular  veinlets  lie  between  the  lobules, 
but  they  differ  from  branches  of  the  portal  vein  in  not  being  accom- 
panied by  a branch  of  the  hepatic  artery  and  bile-duct. 

6.  Transparent  Injections — Liver  of  Pig. — Mount  in  the  same 
way  a transparent  injection  of  the  liver  of  a pig.  P.Y.  blue,  and 
H.Y.  red. 

(a.)  (L)  The  hepatic  veinlet  in  the  centre  of  the  lobule.  If  cut 
transversely,  it  is  circular ; if  obliquely,  oval ; and  if  the  lobule  be 
cut  longitudinally,  it  appears  as  a central  channel  joining  a sub- 
lobular vein.  Trace  outwards  from  this  a radially-arranged  capillary 
network,  with  its  cross-branches,  right  out  to  the  outer  part  of  each 
26  T 


290 


PRACTICAL  HISTOLOGY. 


[xxvi. 


lobule.  Notice  that  the'  shape  of  the  meshwork  of  capillaries  is 
different  at  the  centre  and  periphery.  Between  the  lobules  and 
outside  each  lobule  branches  of  the  portal  vein. 

7.  Liver  of  Rabbit,  Injected  (fig.  280),  a section  with  its 
blood-vessels  injected  with  different  colours,  e.^.,  P.Y.  red  (carmine 
gelatine)  and  H.Y.  blue  (Berlin- blue  gelatine).  Also  a section 
where  all  the  blood-vessels  are  injected  with  a mass  of  one  colour. 
In  a double  injection  the  red  and  blue  masses  do  not  always  occupy 
the  area  corresponding  to  the  blood-vessel  into  which  they  were 
injected,  but  with  care  such  a piece  can  be  found  in  an  injected 
liver.  The  arrangement  of  the  blood-vessels  corresponds  to  that 
seen  in  the  uninjected  specimens,  only  in  the  former  the  blood- 
vessels are  more  prominent  than  the  cells. 

(a.)  (L)  Observe  the  interlobular  veins  round  the  periphery  of 
the  lobule,  the  central  or  intralobular  vein,  and  the  plexus  of 


capillaries  connecting  the  two.  The  capillaries  converge  towards 
the  centre  of  the  lobule,  where  the  meshes  are  more  elongated. 
At  the  periphery  of  the  lobule  the  capillary  meshwork  is  more 
polygonal. 

Yery  instructive  also  are  those  with  only  one  set  of  vessels 
injected,  e.g.^  portal  or  hepatic.  They  serve  to  show  the  capillaries 
belonging  to  each  area  within  a lobule. 

8.  Injected  and  Stained  Section. — A beautiful  preparation  is 
obtained  by  staining  a section  (blue  injection)  with  picro-lithium 
carmine.  In  it  the  vessels  are  blue,  the  cells  yellow,  and  the  nuclei 
of  the  latter  red. 

9.  Bile-Ducts. — Mount  in  balsam  a section  of  a rabbit’s,  or,  better 
still,  a guinea-pig’s  liver  in  which  the  bile-ducts  have  been  injected 
with  a concentrated  watery  solution  of  Berlin-blue.  It  is  very 


XXVI.] 


LIVER. 


291 


difficult  to  get  a perfect  injection,  but  it  is  easy  in  the  guinea-pig  to 
inject  the  large  bile-ducts.  They  are  injected  from  the  common 
bile-duct  after  ligature  of  the  cystic  duct.  Sometimes  it  is  better 
not  to  clamp  the  cystic  duct,  as  after  the  gall-bladder  is  full  it  acts 
as  a reservoir,  and  prevents  too  great  pressure,  causing  extravasations 
of  the  injection  fluid. 

{a.)  (L)  Large  Bile-Ducts. — Numerous  sections  of  these  lying  in 
portal  canals  between  the  lobules.  Each  is  provided  with  a fibrous 
coat  containing  circular,  smooth,  muscular  fibres,  lined  by  columnar 
epithelium.  In  the  walls 
of  the  largest  ducts  are 
mucous  glands,  which 
open  into  the  bile-duct. 

(6.)  (H)  The  bile- 

capillaries  within  the 
lobules  appear  as  a fine 
hexagonal  network  of 
blue  lines  between  the 
surfaces  of  the  hepatic 
cells  (fig.  282).  If  cut 
transversely,  they  appear 
as  mere  blue  specks 
between  adjacent  cells. 

They  are  not  to  be  con- 
founded with  the  blood- 
capillaries,  which  are 
much  wider,  and  are 
arranged  in  a different 
way. 

(c.)  The  columnar  epi- 
thelium lining  the  interlobular  bile-ducts  and  their  fibrous  walls 
(fig.  281). 

10.  Auto-Injection  of  Bile-Ducts. — Place  a piece  of  indigo- 
carmine,  about  the  size  of  a split  pea,  under  the  skin  of  the  fore- 
arm of  a pithed  (brain-destroyed)  frog.  Tie  the  slit  to  prevent  its 
escape.  After  twenty-four  hours  the  whole  frog  will  appear  quite 
blue.  Kill  it,  rapidly  remove  the  liver,  cut  it  in  small  pieces,  and 
place  it  at  once  in  absolute  alcohol,  which  fixes  the  blue  colour. 
After  it  is  hardened,  cut  sections  and  mount  in  balsam. 

(a.)  (H)  The  blood-vessels  are  yellow,  with  gland-tubes  between, 
but  the  bile-capillaries  are  blue.  They  can  be  seen  as  blue  zigzag 
fine  streaks  between  the  cells  when  the  tubes  are  cut  longitudinally, 
and  as  very  small  dots  when  cut  transversely.  A thin  section  gives 
a clear  view  of  the  relation  of  blood-capillaries,  cells,  and  bile 
capillaries. 


Fig.  282. — Lobule  of  Rabbit’s  Liver.  Vessels  and  bile- 
ducts  injected.  P.V.  Portal,  and  H.V.  Hepatic 
vein  ; B.D,  Rile-duct,  and  B.C.  Bile-capillaries. 


2g2 


PRACTICAL  HISTOLOGY. 


[xxvi. 


A good  plan  is  to  clarify  an  unstained  section  with  clove-oil  con- 
taining eosin.  The  cells  are  stained  red,  and  form  a sharp  contrast 
to  the  blue.  A watery  solution  such  as  picro-carmine  cannot  be 
used  to  stain  the  cells,  as  it  rapidly  extracts  the  blue  from  the  bile- 
channels. 

11.  Fresh  Liver-Cells. — Scrape  the  surface  of  a fresh  liver,  and 
observe  in  normal  saline. 

(H)  Observe  the  pale,  nucleated,  polygonal,  or  cubical  faintly- 
granular  cells,  often  containing  small  refractive  oil  globules.  Often 
the  cells  are  broken  up.  There  are  always  many  blood-corpuscles 
in  the  field  (fig.  94). 


ADDITIOI^AL  EXERCISES. 

12.  Connective-Tissue  Stroma. — With  a camel’s- hair  brush  pencil  away  as 
many  as  possible  of  the  hepatic  cells  from  a thin  section  of  any  pro}>erly 
hardened  liver.  The  connective-tissue  network  may  be  afterwards  stained 
with  picro-lithium  carmine  or  eosin. 

13.  Intralobular  Connective  Tissue. — Various  methods  have  been  adopted 
to  show  the  existence  of  connective  tissue  within  the  liver  lobules  and  between 
its  cells.  The  following  methods  reveal  a network  of  black  fibrils  continuous 
with  the  interlobular  tissue,  and  traversing  as  a network  the  substance  of  the 
lobule.  This  method  is  also  useful  for  showing  the  trabeculae  of  the  spleen, 
and  the  fibrillar  structure  in  the  splenic  corpuscles  and  lymph-glands  generally. 

(a.)  Bohm's  Method, 

(1.)  Harden  small  pieces  (.5  cm.  cube)  in  0.5  per  cent,  chromic  acid  (2 
days). 

(2.)  Silver  nitrate  .75  per  cent.  (3  days). 

(3.)  Wash  in  distilled  water,  harden,  cut,  and  mount  in  balsam. 

(5.)  OppeVs  Method  is  available  for  liver  hardened  in  alcohol. 

(1.)  Yellow  chromate  of  potash  10  per  cent,  (i  day). 

(2.)  Silver  nitrate,  large  volume,  .75  per  cent.  (2  days).  Much  silver 
chromate  is  formed,  so  that  the  silver  must  be  frequently 
renewed. 

(3.)  Wash  in  water,  harden  in  alcohol,  sections,  balsam. 

By  either  method  the  liver  cuts  readily  on  freezing,  but  hand  sections,  I 
think,  are  best.  Paraffin  embedding  is  not  to  be  recommended. 

14.  Bile-Capillaries  {Bohm).  Golgi’s  method. 

(1.)  Harden  (3-4  days)  small  pieces  of  the  liver  (.5  cm.  cube)  in 

Bichromate  potash  solution  (3  per  cent.)  . 4 vols. 

Osmic  acid  (i  per  cent.)  ....  i vol. 

(2.)  Place  in  0.75  per  cent,  silver  nitrate  (2  days). 

(3.)  Wash  in  distilled  water,  harden  in  alcohol. 

(4.)  Sections.  Balsam. 


XXVI.] 


LIVER. 


293 


The  bile-capillaries  appear  as  a black  polygonal  network  on  a yellow  ground. 
It  is  rare,  I find,  to  have  the  whole  tliickness  of  the  tissue  equally  well 
stained.  Only  a thin  layer  on  the  surface  shows  the  capillaries  well,  but  the 
result  is  excellent.  I find  that  the  sections  keep  for  a long  time  if  they  are 
mounted  under  a cover-glass. 

15.  Glycogen  in  Liver-Cells. — (a.)  The  animal  must  be  well  fed,  e.g.,  rabbit, 
with  carrots,  and  six  hours  or  so  thereafter  it  is  killed.  As  glycogen  is  soluble 
in,  or  is  at  least  extracted  from,  the  liver  by  water,  small  pieces  of  the  liver 
must  be  hardened  in  alcohol.  In  a section  placed  in  a weak  iodine  solution 
or  Lugol’s  solution  (p.  93),  the  glycogen  granules  in  the  cell  protoplasm 
are  stained  of  a port-wine  colour.  In  sections  of  liver  sometimes  one  sees 
Viicuoles  from  which  the  glycogen  granules  liave  been  washed  out. 

{b. ) Haiden  the  liver  of  a well-fed  frog  in  osmic  a(dd,  make  thin  sections, 
and  irrigate  with  iodine.  The  granules  of  glycogen  in  the  hepatic  protoplasm 
are  stained  brownish. 

16.  Reaction  for  lron{  Tizzoni's). — {a.)  Select  the  liver  of  a young  animal,  and 
harden  it  in  alcohol.  Place  sections  in  the  following  fluid,  which  should  be 
freshly  prepared  ; — 

Water 90  cc. 

Hydrochloric  acid  (25  per  cent.)  . . . • i-5  »» 

Ferricyanide  of  potash  (i  : 12)  . . . 3 ,, 

Mount  in  balsam.  Particles  of  free  iron  are  coloured  blue.  Particles  of  free 
iron  are  seen  in  the  spleen,  liver,  and  kidney  by  this  reaction. 

{b.)  Zaleski?- — Harden  the  liver  in  65  per  cent,  alcohol,  then  in  96  per  cent, 
alcoliol  to  which  a few  dro}>s  of  sulphuretted  hydrogen  are  added.  After  24 
hours  the  iron  granules  assume  a green  colour. 

17.  Injected  Human  Liver. — [a.)  For  a double  injection,  and  in  order  to 
save  injection-mass,  the  plan  recommended  "by  Orth  is  excellent,  viz.,  to  pass 
an  elastic  catheter  as  far  as  it  will  go  into  one  of  the  branches  of  the  portal 
vein,  and  through  it  to  make  the  injection.  The  hepatic  vein  is  treated  in 
the  same  way.  Sections  of  a well-injected  part  may  be  afterwards  stained 
with  picro-lithium-carmine. 

(0.)  In  a way  a natural  injection  of  the  liver  is  obtained  by  hardening  a 
human  liver  which  is  congested,  and  contains  a large  amount  of  blood.  Such 
livers  are  apt  to  show  pigmentation  of  the  liver-cells  and  other  changes  due  to 
disease. 

18.  Pigment  in  Liver. — The  presence  of  pigment  in  the  liver-cells  or  capil- 
laries is  a matter  of  considerable  im[)ortance  in  regard  to  the  question  of  the 
destruction  of  blood-pigment  in  this  organ,  or  whether  the  liver  acts  as  a trap 
for  pigment  already  altered  by  its  passage  through  the  spleen  or  gastro- 
mesenteric  capillaries. ^ 

In  winter  frogs  the  liver  contains  a large  amount  of  black  pigment,  which 
lies  in  the  blood-capillaries. 

19.  Granular  Cells  of  Ehrlich. — Harden  the  liver  (ox,  pig)  in  absolute 
alcohol.  Place  sections  for  24  hours  in  Westphal’s  fluid  (p.  67),  w^ash  in 
absolute  alcohol  (4-6  hours)  until  all  is  clear,  only  the  granular  cells  remaining 
stained.  Balsam.  The  granular  cells  (‘* Mastzellen  ”)  are  reddish  violet, 
and  lie  in  the  interlobular  connective  tissue. 

^ Zeitsch,  f,  Phys.  Chem.,  xiv.  p.  274,  1890. 

2 W.  Hunter,  Brit.  J[fed.  Jour.^  Nov.,  Dec.  1892. 


294 


PRACTICAL  HISTOLOGY. 


[XXVII. 


LESSOR  XXYII. 

TRACHEA— LUNGS— THYROID  GLAND. 

The  trachea,  a fibro-muscular  tube,  the  wall  of  which  contains 
16-20  C-shaped  pieces  of  hyaline  cartilage  held  together  by  a 
fibrous  membrane.  Behind,  the  rings  are  deficient,  and  the  trachea 
is  membranous,  and  there  it  is  strengthened  by  smooth  muscle — 
trachealis  muscle — which  stretches  between  the  ends  of  the  carti- 
lages. It  is  lined  by  a mucous  membrane,  which  is  united  to  the 
outer  fibrous  coat  by  a submucous  coat.  The  mucous  coat  consists 
from  within  outwards  of — (i.)  Stratified,  columnar,  ciliated  epithe- 
lium; (2.)  Basement  membrane;  (3.)  A basis  of  connective  tissue 
with  capillaries,  and  infiltrated  with  adenoid  tissue ; (4.)  A layer  of 
elastic  fibres  arranged  longitudinally.  Outside  this  is  a loose  sub- 
mucous coat,  in  which  lie  the  acini  of  the  glands. 

The  intra-pulmonary  bronchi  are  lined  by  stratified  ciliated  epi- 
thelium resting  on  a basement  membrane.  Outside  this  is  a basis 
of  fibrous  tissue,  with  numerous  longitudinally-arranged  elastic 
fibres,  and  some  adenoid  tissue.  Outside  this,  again,  is  a com- 
pletely circular  layer  of  smooth  muscle — bronchial  muscle.  Then 
follows  the  submucous  coat  with  its  vessels,  glands,  and  in  some 
animals  (cat)  masses  of  adenoid  tissue.  Most  externally  is  the 
fibrous  coat,  in  which  are  embedded  several  pieces  of  hyaline 
cartilage  of  irregular  shape.  As  the  bronchi  pass  into  the  lung, 
they  divide  and  form  smaller  and  smaller  tubes,  until  they  end 
in  terminal  bronchi  or  bronchioles.  Each  bronchiole,  with  thin 
walls,  no  glands  or  cartilage,  and  the  epithelium  cubical  and  non- 
ciliated,  opens  into  several  wider  expanded  parts — infundibula  or 
alveolar  passages — which  are  beset  with  air-cells  or  alveoli.  The 
alveoli  are  spherical  or  polygonal  vesicles,  which  open  by  a wide 
opening  into  the  infundibula ; the  air-vesicles,  however,  do  not  open 
into  each  other.  The  air-cells  are  lined  by  a layer  of  squames — 
large,  flattened,  irregular  plates — with  small  granular  cells  here  and 
there  between  them. 

Blood-Vessels. — The  branches  of  the  pulmonary  artery  accom- 
pany the  bronchial  tubes,  and  finally  terminate  in  a rich  capillary 
plexus  over  and  outside  the  basement  membrane  of  the  air-vesicles. 
The  blood  is  returned  by  the  pulmonary  veins.  The  bronchial 
vessels  seem  to  supply  chiefly  the  connective  tissue  along  the 
bronchi  and  in  the  septa.  Numerous  ganglia  exist  in  the  intra- 
pulmonary  nerves.  Lymphatics  are  numerous. 


xxvil] 


THE  RESPIRATORY  ORGANS. 


295 


THE  RESPIRATORY  ORGANS. 

Methods. — (i.)  Place  small  portions  of  the  trachea  of  a cat  and  the 
human  trachea  in  .2  per  cent,  chromic  acid  (10-14  days)  or  sub- 
limate, and  harden  afterwards  in  gradually  increasing  strengths  of 
alcohol. 

(ii.)  Fill  the  lungs  of  a cat  with  .2  per  cent,  chromic  acid,  and 
after  closing  the  trachea  suspend  them  in  a large  quantity  of  the 
same  fluid.  After  two  days  cut  them  into  small  portions  and  keep 
them  (14-20  days)  in  fresh  chromic  acid  solution  and  complete  the 
hardening  in  alcohol. 

(iii.)  Harden  small  parts  of  the  human  lung — as  fresh  as  j)Ossihle 
— in  the  same  fluids. 

(iv.)  Fix  the  trachea  in  Flemming’s  fluid,  harden  in  alcohol,  and 
stain  with  safranin.  Admirable  for  epithelium. 

Trachea. — Make  transverse  sections  across  the  trachea  of  a cat, 
and  also  longitudinal  vertical  sections,  so  as  to  include  the  cartilage 
of  two  or  three  rings.  This 
is  best  done  by  staining  in 
bulk  and  cutting  in  paraffin. 

If  done  by  freezing,  stain 
T.S.  and  L.S.  sections  in 
hsematoxylin,  and  mount  in 
balsam ; others  in  picro-car- 
mine,  and  mount  in  Farrant’s 
solution. 

1.  T.S.  Trachea  (fig.  283). 

(a.)  (L)  Observe  internally 
the  mucous  coat,  outside  it 
the  submucous  coat,  and 
external  to  this  an  incom- 
plete ring  of  hyaline  cartilage 
embedded  in  the  outer  or 
fibrous  coat.  The  fibrous  coat  becomes  continuous  with  the  sub- 
mucous coat. 

(b.)  (H)  The  fibrous  coat  of  connective  tissue,  and  embedded 
in  it  an  incomplete  ring  of  hyaline  cartilage.  Notice  the  arrange- 
ment of  the  cartilage  cells  in  the  latter.  The  ring  is  deficient 
posteriorly,  but  bridging  over  the  gap  and  extending  between  the 
ends  of  the  cartilage  there  is  a transverse  band  of  smooth  muscle, 
the  trachealis  muscle.  Outside  it  are  muscular  fibres  cut  trans- 
versely. The  coats  just  inside  the  muscle  are  apt  to  be  thrown 
into  folds  in  a trachea  detached  from  all  its  surroundings.  Sections 
of  nerves — perhaps  with  ganglionic  cells — may  be  seen  near  the 
muscle. 


Fig.  283.— T.S.  Trachea  of  Kitten,  x 15. 


296 


PRACTICAL  HISTOLOGY, 


[XXVII, 


(c.)  The  submucous  coat,  composed  of  more  open  connective 

In  it  are  the  acini  of 
mucous  glands;  but  as 
the  glands  are  more 
abundant  in  the  spaces 
between  the  cartilages, 
their  distribution  is 
better  seen  in  a longi- 
tudinal section.  It  is 
rare  to  find  a duct,  as 
they  pierce  the  mucous 
coat  obliquely,  and 
open  on  its  inner  sur- 
face. 

(d.)  The  mucous 
coat,  composed  of 
fibrous  tissue  covered 
by  stratified  ciliated  epithelium.  Under  the  epithelium  is  a longi- 
tudinal layer  of  elastic  fibres,  which  are  therefore  cut  across  trans- 
versely. Within  this,  a basis  of  connective  tissue  infiltrated  with 
• adenoid  tissue,  and  internal  to  this  again  a structureless  basement 


Fig.  285. — T.S.  Intra-Pul  m on  ary  Bronchus  of  Cat.  PA  and  PV.  Pulmonary  artery  and 
vein;  bv.  Bronchial  vein;  V,  Air-vesicles. 

membrane,  best  seen  in  very  thin  sections,  and  best  of  all  in  a 
human  trachea.  Resting  on  the  basement  membrane  is  the  ciliated 
einihelium.  It  occurs  in  several  layers,  but  only  the  superficial 
layer  of  cells  is  ciliated.  In  the  lower  layers  of  cells — three  or  four 


tissue,  and  continuous  with  the  former. 


Fig.  284.— L.S.  Mucous  Membrane  of  Trachea  of  Cat. 


xxvil] 


THE  RESPIRATORY  ORGANS. 


297 


layers — some  are  pear-shaped  and  the  lowest  more  oval.  Yery 
frequently  a thin  layer  of  mucus  is  adherent  to  the  cilia.  The 
arrangement  of  the  cells  of  the  mucous  layer  is  the  same  as  in  the 
bronchus  (fig.  286). 

2.  L.S.  Trachea  (fig.  284)  (L  and  H).^ — Compare  these  with  the 
previous  section.  The  coats  are  the  same,  but  several  oval  pieces 
of  hyaline  cartilage  are  seen  one  after  the  other  embedded  in  the 
fibrous  coat.  The  elastic  fibres  are  now  seen  arranged  longitudi- 
nally. The  acini  of  the  mucous  glands  are  most  numerous  in  the 
interspaces  between  the  cartilages,  and  amongst  the  acini  may  be 
seen  leucocytes  and  some  adenoid  tissue.  If  a gland-duct  be  found, 
it  opens  by  a funnel-shaped  expansion  on  the  free  surface  of  the 
mucous  membrane.  If  it  be  desired  to  study  the  glands,  use 
haematoxylin  and  eosin  as  stains. 

3.  T.S.  Human  Trachea  (L  and  H).  — Observe  the  general 
similarity  to  the  previous  prepara- 
tion. Here,  however,  the  glands 
are  well-developed ; the  basement 
membrane  is  well  marked,  and  in 
picro-carmine  specimens  is  stained 
red.  Some  of  the  superficial  epi- 
thelial cells  are  apt  to  be  detached. 

4.  T.S.  Intra-Pulmonary  Bron- 
chus.— (a.)  (L)  Observe  the  -fibrous 
coat,  and  outside  it  the  vesicular 
tissue  of  the  lung.  In  the  fibrous 
coat  — two  or  three  — pieces  of 
hyaline  cartilage.  The  submucous 
coat,  with  its  glands  (fig.  285). 

(6.)  Inside  this  a complete  ring 
of  smooth  muscle — bronchial  muscle 
— perforated  here  and  there  by  the 
ducts  of  the  glands.  Immediately  ^iq,  286 
inside  this,  in  the  mucous  coat, 
several  layers  of  longitudinal  elastic 
fibres  cut  transversely.  Most  in- 
ternal, ciliated  epithelium,  like 
that  lining  the  trachea,  and  resting 
on  a basement  membrane.  The  mucous  membrane  is  frequently 
thrown  into  ridges  or  folds. 

(c.)  In  the  fibrous  coat,  external  to  the  cartilages,  search  for 
sections  of  two  large  vessels — the  pulmonary  artery  and  vein — and 
of  small  branches — the  bronchial  vessels.  Also  several  sections  of 
nerves;  in  the  course  of  some  of  them  may  be  found  ganglionic 
cells. 


T.S.  Mucous  Meml)rane  of 
Human  Bronchus,  a.  Mucus ; h. 
Ciliated  cells ; 0.  Deep  cells;  d.  Base- 
ment membrane  ; e.  Longitudinal 
elastic  fibres  ; /.  Bronchial  muscle  ; 
g.  Connective  tissue,  with  leucocytes 
and  pigment. 


27 


298 


PRACTICAL  HISTOLOGY. 


[XXVII. 

(6?.)  (H)  study  specially  the  mucous  membrane.  ]^ote  the 
epithelium,  basement  membrane,  and  the  longitudinal  layer  of 
elastic  fibres  under  it  (fig.  286). 

5.  Vesicular  Structure  of  the  Lungs. — Make  sections  of  a 
lung  hardened  by  the  freezing  method.  Let  the  sections  be  cut  so 
as  to  include  the  pleura  and  subjacent  lung.  Stain  in  haematoxylin, 
and  mount  in  balsam. 

(a.)  (L)  Observe  the  pleura,  with  its  two  layers,  the  deeper 


Fig.  287. — V.S.  Human  Lung.  p.  Pleura;  a.  Epithelium  of  a bronchus  ; h.  Blood-vessel; 
c.  Pulmonary  vein ; e.  Interlobular  septum,  continuous  with  deep  layer  of  pleura; 
V.  Air- vesicles  ( x 50  and  reduced  ^). 

layer  sending  fine  septa — interlobular  septa — into  the  lung  between 
its  alveoli  (fig.  287). 

{h. ) The  alveoli  or  air-cells  cut  in  every  direction ; some  appear 
as  an  open  network,  and  others  with  a base.  The  outline  of  the 
alveoli  may  be  somewhat  irregular,  according  to  the  extent  of  dis- 
tension of  the  lung.  Here  and  there,  between  groups  of  alveoli, 
may  be  seen  a wide  passage — the  infundibulum. 


XXVII  ] 


THE  RESPIRATORY  ORGANS. 


299 


(c.)  (H)  In  each  alveolus  stained  oval  nuclei,  belonging  to  the 
squamous  epithelium  lining  it,  but  the  outline  of  the  squames  them- 
selves cannot  be  seen.  Sections  of  the  capillaries  on  the  wall  of  the 
alveolus  and  their  nuclei  stained. 

In  the  thin  walls,  separating  ad- 
joining alveoli,  fine  elastic  fibres. 

6.  Eespiratory  Epithelium 
of  the  Alveoli. — (i.)  Remove 
the  lungs  from  a young  kitten, 
and  fill  them  with  a ^ per  cent, 
solution  of  silver  nitrate.  Run 
in  the  fluid  from  a pipette  pro- 
vided with  a bulb  on  its  stem. 

Pump  the  lungs  from  time  to 
time  to  remove  as  much  air  as 
possible.  After  half  an  hour 
replace  as  much  as  possible  of 
the  silver  solution  by  means  of 
alcohol.  Tie  the  trachea,  and 
suspend  the  lungs  in  alcohol  till 
they  are  hardened.  Cut  sections 
by  freezing,  mount  in  Tarrant’s 
solution,  and  expose  the  sections 
to  light.  They  become  brown. 

Sections  can  be  stained  in 
hsematoxylin  or  picro-carmine. 

(ii.)  Fill  the  lungs  with  .25  per  cent.  AgXOg,  and  suspend  them 
in  .5  per  cent.  AgNOg  (1-2  hours).  Dihusion  takes  place.  Cut 
into  pieces  and  place  in  alcohol  (80  per  cent.). 

(H)  Select  an  alveolus  which  is  so  divided  as  to  have  a base,  and 
observe  the  silver  lines  showing  the  boundary-lines  of  the  squamous 
epithelium  which  lines  it.  At  the  junction-points  of  some  of  these 
are  groups  of  two  or  three  small,  polyhedral,  granular  cells  stained 
deep  brown  (fig.  288). 

7.  Blood-Vessels  of  a Mammalian 
Lung. — Mount  in  balsam  sections  of  an 
injected  lung. 

(L  and  H)  Observe  the  alveoli,  each 
beset  with  a dense  plexus  of  capillaries 
(fig.  289),  and  especially  where  the  edge 
of  an  alveolus  is  seen,  the  wavy  course 
of  the  capillaries  passing  from  one  side 
to  the  other  of  the  inter- vesicular  septa. 

Search  for  the  termination  of  a branch  of 
the  pulmonary  artery  and  the  commencement  of  the  pulmonary  vein. 


Fig.  288.  — Alveoli  oi  Lung  of  Kitten, 
Silvered,  a,  b.  Squames ; d.  Granular 
cells  ; c.  Young  epithelium-cell ; e. 
Alveolar  wall. 


Fig.  289. — Capillaries  of  Human 
Lung,  Injected,  x 90. 


300 


PRACTICAL  HISTOLOGY. 


[XXVIL 


8.  Fresh  Lung. — Tease  a small  piece  of  lung  in  normal  saline. 
It  is  difficult  to  get  rid  of  all  the  air,  but  this  may  be  done  by 
beating  the  tissue  with  a needle. 

(H)  Observe  the  large  number  of  fine  elastic  fibres,  which 
branch  and  anastomose.  They  can  be  rendered  more  evident  by 
running  in  dilute  caustic  potash  (2  percent.)  under  the  cover.  This 

destroys  to  a large  extent  the  other 
elements. 

9.  Dried  Lung. — (i.)  With  a dry 
razor  make  thin  sections  of  a dried 
and  distended  lung.  Examine  the 
section  in  water,  taking  care  that 
it  does  not  curl  up,  which  it  readily 
does.  Get  rid  of  the  air-bubbles  by 
pressing  on  the  section  with  a needle. 

(ii.)  Make  rather  thick  sections 
parallel  to  the  pleura,  and  examine 
them  by  reflected  light.  A very  good 
idea  of  the  air-vesicles  is  thus  ob- 
tained. 

(a.)  (L)  Observe  the  air-vesicles, 
and  the  thin  partitions  between  them.  Also  sections  of  the 
infundibula  or  alveolar  passages.  Connective-tissue  septa  may 
be  seen.  Of  course  the  finer  details  of  structure  cannot  be  made 
out  (fig.  290). 

10.  Foetal  Lung. — This  serves  very  well  for  showing  that  a lung 


Pig.  290.— T.S.  Dried  Lung.  a.  Vesi- 
cles; 1.  Infundibula. 


Fig.  291. — T.S.  Foetal  Lung,  showing  a Bronchus  Terminating  in  Air-Vesicles,  X75 
B.  Epithelium  lining  an  alveolus,  x 300.  Muller’s  fluid  and  hsematoxylin. 


is  made  up  of  lobules.  Harden  in  Muller’s  fluid  (14  days)  the 
lungs  from  a human  foetus  (12  cm.  in  length).  Cut  sections  and 


XXVII.]  THYROID  GLAND.  3OI 

stain  them  either  in  hsematoxylin  or  picro-carmine.  Stain  in  bulk 
and  cut  in  paraffin. 

(a.)  (L)  Observe  the  pleura  sending  well-marked  septa  into  the 
lung,  thus  defining  the  lobules,  which  are  polygonal,  about  i mm. 
in  diameter,  and  separated  from  each  other  by  connective  tissue. 

(h.)  In  each  lobule  sections  of  bronchi,  which  can  be  seen 
occasionally  to  terminate  in’ several  vesicles,  thus  presenting  a very 
gland-like  arrangement  (fig.  291).  Many  of  the  alveoli  are  cut 
across,  and  appear  like  sections  of  tubes  lined  by  columnar  epithe- 
lium. There  is  much  embryonic  connective  tissue  between  the 
alveoli. 

(H)  Select  an  alveolus,  and  note  that  it  is  lined  by  a layer  of 
low,  columnar,  granular  epithelium,  while  individual  alveoli  are 
separated  from  each  other  by  much  embryonic  connective  tissue, 
with  numerous  cells,  and  as  yet  few  or  no  elastic  fibres. 

THE  THYROID  GLAND. 

Methods. — (i.)  Harden  pieces  of  the  human  thyroid,  or  the 
complete  thyroid  of  a cat  or  dog,  in  Muller’s  fluid  (3  weeks)  and 
then  in  alcohol.  Sections  are  stained  with  haematoxylin  and 
mounted  in  balsam. 

Or  stain  in  bulk  and 
cut  in  paraffin. 

(ii.)  Fix  in  Flem- 
ming’s fluid  (1-3 
hours),  and  stain — 
wash  thoroughly, 
alcohol — with  Ehr- 
lich-Biondi  fluid,  or 
stain  with  Heiden- 
hain’s  logwood. 

11.  T.S.  Thyroid 
Gland. — (a.)  (L) 

Composed  of  small 
polygonal  lobules 
united  to  each  other 
by  loose  connective 
tissue.  In  each  lob- 
ule a large  number 
of  completely  closed  acini  held  together  by  loose  connective  tissue. 

{h.)  (H)  The  spherical  acini  (fig.  292),  bounded  by  a basement 
membrane,  are  lined  by  a single  layer  of  low  cubical  epithelium,  and 
contain  a homogeneous  fluid.  This  fluid,  however,  is  often  of  a 


Fig.  292.— T.S.  Thyroid  Gland,  a.  Closed  vesicle. 


302 


PRACTICAL  HISTOLOGY. 


[XXVII. 


colloid  nature,  and  is  then  a pathological  product.  It  is  stained  by 
logwood.  Kumerous  sections  of  blood-  and  lymph-vessels  outside 
the  basement  membrane  of  the  acini. 

12.  Injected  Thyroid  (thickish  section  in  halsam). — (L)  Numer- 
ous large  vessels  in  the  connective  tissue,  with  a plexus  of  capil- 
laries over  the  acini,  but  outside  their  basement  membrane. 


ADDITIONAL  EXERCISES. 


13.  Lung  of  Newt. — (i. ) This  is  an  elongated  sac,  and  is  of  comparatively 
simple  structure.  Fill  a lung  with  gold  chloride  (.5  per  cent.),  and  suspend 
it  in  a few  cc.  of  the  same  Huid  for  twenty  minutes.  Reduce  the  gold  by 
exposure  to  sunlight  in  water  feebly  acidulated  with  acetic  acid.  Mount  a 
portion  of  the  thin  wall  in  glycerine. 

{a.)  (L)  Observe  islands  or  small  groups  of  epithelial  cells.  They  lie  in  the 
intercapillary  spaces.  The  capillaries  are  wide,  and  form  an  anastomosing 
network.  Outside  this  capillary  layer  is  a layer  of  smooth  muscle,  and  one  of 
fibrous  tissue. 

(ii.)  If  the  gold  chloride  be  reduced  by  formic  acid  (25  per  cent,  in  the  dark), 
the  epithelium  lining  the  lung  is  shed,  and  then  the  nerves  to  the  lung — with 
many  ganglia  in  their  course — can  be  seen. 

14.  Lung  of  Frog. — (i.)  Fill  a lung  with  dilute  alcohol,  suspend  it  in  the 
same  fluid  (twenty-four  hours),  lay  oj^en  the  lung  and  pencil  away  the  inner 
lining  epithelium  and  mount  in  Farrant’s  solution. 

{a.)  (L)  Observe  the  large,  coarse,  but  short  primary  septa,  which  project 
inwards  from  the  wall  of  the  lung  towards  the  large  central  cavity.  From 
them  secondary  septa  pass  to  form  a trabecular  arrangement,  thus  giving  the 
interior  of  the  lung  a honeycomb-looking  appearance.  The  trabeculae  consist 
chiefly  of  smooth  muscle. 

(ii.)  The  nerves  of  the  frog’s  lung  are  readily  demonstrated  by  the  gold 
chloride  formic  acid  method  and  the  methylene-blue  method.  The  numerous 
ganglionic  cells  in  the  course  of  the  nerves  have  a straight  and  a spiral 
process. 

15.  Elastic  Fibres  in  Trachea  and  Lung. — (i.)  Stain  a longitudinal  section 
of  the  har  dened  human  trachea  according  to  the  method  described  in  Lesson 
X.  10.  The  elastic  fibres  become  black. 

(ii.)  Use  the  safranin  method  (Lesson  X.  9).  This  shows  beautifully  the 
arrangement  of  the  fibres,  now  of  a purplish  or  black  tint. 

16.  Elastic  Fibres  in  the  Lung. — (i.)  I have  devised  the  following  two 
methods,  which  give  good  results : —Make  sections  of  a dried  and  distended 
lung,  stain  a section  in  dilute  magenta,  and  allow  the  section  to  dry  com- 
pletely on  a slide  ; add  balsam  and  cover.  The  elastic  fibres  are  red,  and  their 
arrangement  can  be  seen  with  the  utmost  distinctness. 

(ii. ) Or.  stain  a section  in  methyl-violet,  and  clarify  it  wit  1 the  aniiine-oil 
and  xylol  mixture  (p.  123). 


XXVIII.] 


KlDi^EY, 


303 


LESSOR  XXYIII. 

KIDNEY— URETER— BLADDER 


KIDNEY. 

If  a kidney  be  divided  longitudinally,  one  distinguishes  a cortical 
and  a medullary  part,  the  latter  consisting  in  different  animals  of 
one  or  more  pyramidal 


portions — the  pyramids 
of  Malpighi  — whose 
apices  project  into  the 
pelvis  of  the  kidney, 
while  their  bases  are 
surrounded  by  cortical 
substances.  The  medul- 
lary portion  is  subdi- 
vided into  the  boun- 
dary or  intermediate 
zone  and  the  papillary 
portion  (fig.  2 93).  The 
kidney  is  covered  by  a 
loosely  adherent  cap- 
sule. It  is  a compound 
tubular  gland,  consist- 
ing of  numerous  urini- 
ferous  tubules  closely 
packed  together  with 
very  little  connective 
tissue  between  them, 
the  connective  tissue 
carrying  the  blood-ves- 
sels, lymphatics,  and 
nerves.  The  urini  - 
ferous  tubules  pursue  a 
straight  course  in  the 
medulla,  but  they  ex- 
hibit a contorted  or 
convoluted  arrangement 
straight  tubules- 


Boundary  or 
J '>marginal 
zone. 


Fig.  293. — L.S.  of  a Pyramid  of  Malpighi.  PF.  Pyramids 
of  Ferrein ; Branch  of  renal  artery  with  an  in- 
terlobular artery;  RV,  Lumen  of  a renal  vein  re- 
ceiving an  interlobular  vein ; VR.  Vasa  recta;  PA. 
Apex  of  a renal  papilla ; hb.  Embrace  the  bases  of 
the  lobules. 


in  the  cortex,  although  bundles  of 
-the  medullary  rays  or  pyramids  of  Ferrein— 
pass  into  the  cortex  from  the  medulla  (fig.  293).  Each  uriniferous 


304 


PRACTICAL  HISTOLOGY. 


[XXVIII. 


tubule  consists  of  a basement  membrane  lined  by  a single  layer  of 
epithelium.  The  tubules  alter  their  character  and  course  in  dif- 
ferent parts  of  the  organ.  The  tubules  begin  in  the  cortex  in  a 


Sub-cap- 
sular layer. 


12.  Junc- 
tional tu- 
bule. 

II.  Convolu- 
ted tubule, 
lo.  Irrejju- 
lar  tubule. 

A Cortex. 


3.  Proximal 
convoluted 
tubule. 

p.  Ascend- 
ing tubule. 
2.  Neck. 

4.  Spiral 
tubule. 

I.  Capsule 
and  glome- 
rulus. 


' 8.  Spiral  part  of 
; ascending  limb. 


(u 


5 


! B.  Boundary  Zone. 


7 and  8.  Ascending 
part  of  Henle’s  loop. 


15.  Discharging 
tubule. 


5.  Descending  limb  ' 
of  Henle’s  tubule. 


< 


6.  Henle’s  loop. 


VC. 


Papillary  Zone. 


Fig.  294. — Diagram  of  the  Course  of  Two  TJriniferous  Tubules. 


globular  dilatation — the  Malpighian  capsule — which  encloses  a 
tuft  of  vessels — the  glomerulus  (fig.  294).  The  capsule  leads  into 
a narrow  neck,  which  passes  into  the  first  or  proximal  convoluted 


XXVIII.] 


KIDNEY. 


305 


tubule,  which  in  turn  is  continued  into  the  spiral  tubule.  After 
this  it  narrows  suddenly  and  runs  into  the  medulla  as  the  descend- 
ing tubule  of  Henle,  where  it  forms  a narrow  loop — loop  of  Henle — 
and  passes  in  the  reverse  direction  towards  the  cortex  as  the  ascend- 
ing tubule  of  Henle.  In  the  cortex  it  has  a zigzag  course — irregular 
tubule — and  again  becomes  wider  and  convoluted — second  or  distal 
convoluted  tubule — which  leads  into  a straight  tube — ^junctional 
tubule — which  joins  a straight  or  collecting  tubule.  This  passes 


Tabular  Vieiv  (after  Schafer)  of  the  Parts,  Situation  and  Epithelium 
of  a Uriniferous  Tubule, 


Portion  of  Tubule. 

Kind  of  Epithelium  (always  in  a 
single  layer). 

Situation  of  Tubule. 

I.  Capsule  . . . 

Flattened,  reflected  over 
glomerulus 

Labyrinth  of  cortex. 

2.  First  convoluted 
tube 

Cubical,  rodded  cells,  which 
interlock 

a 

>> 

3.  Spiral  tube  . . 

Like  last,  but  ‘‘rods”  very 
distinct 

Medullary  ray  of 
C'Ttex. 

4.  Descending  limb 
of  Henle’s  tube 

Clear  flattened  cells 

Boundary  zone  and 
partly  in  papillary 
zone. 

5.  Loop  of  Henle  . 

Papillary  zone  of 
medulla. 

6.  Ascending  tube 
of  Henle 

Cubical,  rodded,  sometimes 
imbricated 

Medulla  and  medul- 
lary ray  of  cortex. 

7.  Irregular  zigzag 
tu ' >e 

Cells  cubical,  very  “rodded,” 
lumen  small 

Labyrinth  of  cortex. 

8.  Second  convoluted 
tube 

Like  those  of  (2),  but  longer 
and  more  refractive 

>> 

>» 

9.  Junctional  tube . 

Clear,  flattened,  cubical  cells 

Labyrinth  passing  to 
medullary  ray. 

10.  Straight  or  col- 
lecting tube 

Clear  cubical  and  columnar 
cells 

Medullary  ray  and 
medulla. 

j 1 1.  Duct  of  Bellini  . 

Clear  columnar  cells 

Opens  at  apex  of 
papilla. 

straight  through  the  cortex  and  medulla,  receiving  other  similar 
tubes  as  it  goes ; and  becoming  wider,  it  opens  as  a discharging 
tubule  (duct  of  Bellini)  cn  the  apex  of  a Malpighian  pyramid.  The 
tubes  are  lined  throughout  by  a single  layer  of  epithelium,  which, 
however,  changes  its  characters  in  the  different  parts  of  the  tube. 
The  Malpighian  capsule  is  a globular  expansion  (200  p),  composed  of 
a basement  membrane  lined  by  a single  layer  of  squamous  epi- 
theliumc  In  the  convoluted  tubules,  proximal  and  distal,  the  epithe- 

U 


3o6 


PRACTICAL  HISTOLOGY. 


[XXVIII. 


lial  cells  are  somewhat  cubical,  but  their  outlines  are  not  well 
defined.  They  each  contain  a spherical  nucleus,  and  their  proto- 
plasm is  “ rodded,”  especially  at  the  outer  part.  In  the  spiral 
tahule  the  cells  are  not  unlike  those  of  the  convoluted  tubule,  but 
they  are  not  so  tall,  and  therefore  leave  a more  distinct  lumen,  and 
they  are  not  so  markedly  “rodded.’^  In  the  descending  part  of  the 
loop  and  the  loop  itself — very  narrow”  (10-15  /^) — 
clear  and  flattened,  with  a bulging  opposite  the  nucleus,  and 
tliese  projections  alternate  with  those  on  the  opposite  side  of 
the  tubule.  The  ascending  limb  (30  wide)  has  somewhat 
cubical  cells,  which  leave  a regular  lumen.  They  are  striated, 
and  often  present  an  imbricate  arrangement.  The  irregular 
zigzag  tubule  bends  on  itself  with  sharp  angles,  and  is  wide, 
with  an  irregular  lumen.  Its  cells  stain  deeply  with  staining 
reagents,  and  are  conspicuously  striated  in  their  outer  part.  The 
second  or  distal  convoluted  tubule  is  like  the  proximal.  The  junc- 
tional and  collecting  tubules  are  lined  by  low,  columnar,  clear,  trans- 
parent cells  with  small  nuclei.  The  cells  do  not  stain  readily. 
In  the  discharging  tubules  the  cells  have  the  same  general  character, 
but  they  are  taller  and  more  columnar.  Fig.  294  shows  the  general 
arrangement  of  the  tubules,  and  from  it  it  is  easy  to  see  in  what 
part  of  the  kidney  each  kind  of  tubule  is  placed. 

Blood-Vessels. — The  renal  artery  enters  the  kidney,  splits  into 
liranches  which  run  towards  the  cortex,  and  at  the  junction  of  the 
cortex  and  medulla  form  incomplete  arterial  arches  (fig.  293). 
From  these  arches  arise  the  radiate  or  interlobular  arteries,  run- 
ning in  the  cortex  between  two  medullary  rays  in  a radial  direction 
towards  the  surface.  They  give  off  at  intervals  on  all  sides  short, 
slightly- curved  vessels — vasa  afferentia — which  run  without  branch- 
ing to  end  within  the  Malpighian  capsules,  and  there  form  the 
glomeruli.  The  vas  efferens  comes  out  of  the  Malpighian  capsule 
close  to  where  the  afferent  vessel  enters  it  and  at  the  pole  opposite 
to  the  origin  of  the  uriniferous  tubule.  The  efferent  vessel  splits 
up  into  capillaries,  which  ramify  amongst  the  tubules  of  the  cortex. 
The  blood  is  returned  from  the  cortex  by  interlobular  veins  (fig. 
293),  which  run  alongside  of  the  corresponding  arteries.  The 
medulla  is  supi)lied  by  leashes  of  vessels — vasa  recta — w^hich  for 
the  most  part  proceed  from  the  arterial  arches.  The  vasa  recta  are 
pencils  of  arterioles  (10-15),  splitting  up  into  capillaries  which 
ramify  between  the  tubules  of  the  medulla ; the  medulla,  however, 
is  not  so  vascular  as  the  cortex.  The  blood  is  returned  by  corre- 
sponding veins.  The  connective  tissue  is  very  scanty  in  the  cortex, 
but  abundant  in  and  near  the  apices  of  the  Malpighian  pyramids. 

Methods. — (i.)  Harden  small  pieces  in  a 2 per  cent,  solution  of 
potassic  bichromate  (18-20  days)  or  MiilleFs  fluid.  Corrosive 


XXVIII.] 


KIDNEY. 


307 


sublimate  is  also  very  good,  and  so  is  alcohol.  The  pieces  should 
not  be  large,  and  should  be  cut  according  as  a longitudinal  or  trans- 
verse section  is  desired.  Stain  and  cut  in  bulk  in  paraffin.  In  all 
cases  the  pieces  should  include  both  the  cortical  and  medullary 
portions,  and  should  not  be  more  than  half-an-inch  in  thickness. 

(ii.)  Place  small  pieces  in  boiling  water  and  then  complete  the 
hardening  in  Muller’s  fluid  or  alcohol. 

(iii.)  To  fix  the  epithelium  use  Flemming’s  fluid,  e.g.^  for  mouse’s 
kidney. 

Make  radial  sections  from  the  cortex  to  the  apex  of  a Malpighian 
pyramid.  For  a general  view  stain  a section  in  hsematoxylin  and 
mount  it  in  balsam.  Another  one  should  be  stained  in  picro- 
carmine  or  picro-lithio-carmine  and  mounted  in  Farrant’s  solution. 
For  unstained  sections,  steeping  them  for  twenty -four  hours  in 
I per  cent,  osmic  acid  before  mounting  them  in  Farrant’s  solution 
is  excellent.  The  best  way  is  to  begin  with  a section  of  the  entire 
kidney  of  a small  animal,  such  as  a mouse,  rat,  or  guinea-pig.  In 
this  way  a good  view  is  got  of  the  entire  organ  in  section. 

1.  Radial  Section  of  Kidney  of  a small  mammal. 

(a.)  (L)  Observe  the  capsule  ; it  is  thin  and  apt  to  fall  oft ; the 
cortical  and  medullary  portions  of  the  parenchyma.  The  medulla, 
composed  of  straight  tubules  of  different  sizes,  and  running  radially 
from  the  pelvis  of  the  kidney  outwards. 

(h.)  Trace  some  of  the  straight  tubules  outwards  through  the 
intermediate  layer,  in  bundles — the  pyramids  of  Ferrein  or  medul- 
lary rays — into  the  cortical  layer  (fig.  293,  FF).  Many  medullary 
rays  pass  from  a Malpighian  pyramid,  and  they  run  radially  out- 
wards in  the  cortex  nearly  to  its  outer  part — although  they  do  not 
reach  the  surface — becoming  narrower  as  they  are  traced  outwards. 

(c.)  In  the  cortex,  between  every  two  medullary  rays,  are  con- 
voluted tubes,  twisted  and  cut  in  every  direction,  and  two  rows  of 
glomeruli,  enclosed  in  their  capsules — the  labyrinth.  Here  and 
there  a glomerulus  may  have  fallen  out,  and  the  space  it  occupied 
be  left  as  a round  aperture.  The  regular  arrangement  of  the 
glomeruli  will  only  be  seen  provided  the  section  runs  parallel  to  the 
course  of  the  medullary  rays.  The  glomeruli  are  confined  to  the 
cortex,  but  none  of  them  reach  quite  to  the  capsule. 

{(L)  (H)  In  the  cortex,  the  Malpighian  capsules,  each  enclosing 
a tuft  of  capillaries  or  glomerulus.  They  consist  of  a structureless 
membrane  lined  by  a single  layer  of  squames.  The  oval,  flattened 
nuclei  of  the  latter  are  seen  lying  on  the  inner  surface  of  the  cap- 
sule. Within  each  capsule  a cluster  of  capillaries— glomerulus — 
arranged  in  several  groups.  Although  the  squamous  lining  of  the 
capsule  is  reflected  over  the  capillaries,  it  is  not  easy  to  distinguish 
the  nuclei  of  these  cells  from  the  very  numerous  nuclei  of  the 


3o8 


PRACTICAL  HISTOLOGY. 


[XXVIII. 


FiGo  295.— Glomerulus  and  Sections  of  Con- 
voluted Tubules  of  a Kidney,  x 300, 


capillaries  (fig.  295).  The  basement  membrane  of  the  capsule  is 
continuous  by  a narrow  neck  with  the  basement  membrane  of  a 
convoluted  tubule,  but  it  is  only  in  the  rare  case  where  the  section 

cuts  the  capsule  at  this  level  that 
this  connection  is  seen.  This  con- 
nection must  usually  be  made  out 
on  isolated  tubules,  although  some- 
times it  is  seen  in  sections  of  a 
mouse’s  kidney. 

{e.)  The  convoluted  tubules, 
with  a sinuous  or  twisted  course, 
cut  some  longitudinally  and  others 
transversely.  Each  tube  is  lined 
by  a single  layer  of  cells,  the  indi- 
vidual cells  not  sharply  mapped  off 
from  each  other,  and  leaving  a 
small  lumen  in  the  centre.  The 
outer  part  of  each  cell  is  striated 
or  “ rodded,’’  and  near  its  centre 
it  contains  a spheroidal  nucleus 
(fig.  296). 

(/.)  Irregular  Tubules. — Here 
and  there  in  the  cortex  may  be  seen  zigzag  portions  of  tubules 
of  unequal  width,  and  usually  more  deeply  stained  than  the  rest, 

and  the  epithelium  dis- 
tinctly rodded  (fig.  297). 

{g.)  Note  the  small 
amount  of  connective 
tissue  between  the  tub- 
ules of  the  cortex.  It 
is  best  seen  just  outside 
the  Malpighian  capsules. 
FiGo  296. — Rodded  Epithelium  of  a Convoluted  Tubule.  jg  far  more  abundant 

Ammonium  chromate.  . , i t 1 

in  the  medulla. 

Qi.)  In  the  medulla,  the  straight  tubes,  which  are  largest  and 
widest  where  they  are  about  to  open  on  the  apex  of  a Malpighian 

pyramid — discharging  tubules — but 
it  is  not  so  easy  to  get  a section 
showing  this,  as  from  their  radial 
arrangement  they  are  apt  to  be  cut 
_ ^ T 1 rp  u # obliquely.  The  collecting  and  dis- 

FlG.  297.— Irregular  Tubule,  Kidney  of  , ^ ^ i i 

Dog.  Muller’s  fluid.  charging  tubes  have  a wide  lumen, 

and  are  lined  by  a layer  of  clear 
columnar  cells  with  ovoid  nuclei.  Trace  the  straight  tubules  out- 
wards towards  the  surface  ^ they  become  smaller,  still  their  lumen 


xxviil] 


KIDNEY. 


309 


remains  distinct,  and  they  are  lined  by  clear  nucleated  short 
columnar  or  cubical  epithelium. 

(h)  In  the  medullary  rays  in  the  intermediate  layer  very  narrow 
tubes — the  descending  portion  of  the  looped  tubule  of  Henle  (fig. 
300),  not  unlike  fine  capillaries — may  be  seen,  and  also  the  wider, 
more  deeply  stained  ascending  portion  of  the  same  tubule. 

2.  T.S.  of  the  Apex  of  a Malpighian  {HcBmatoxylin 

and  Balsam). 

(a.)  (L  and  H)  Observe  the  large  amount  of  connective  tissue 
between  the  tubules.  The  large  collecting  tubules  are  cut  across, 
so  that  their  large  lumina  and  the 
clear  columnar  epithelial  cells  lining 
them  are  distinctly  seen  (fig.  298). 

Not  infrequently  the  epithelium 
falls  out,  and  then  the  connective 
tissue  appears  as  a network  with 
round  or  oval  holes. 

3.  T.S.  Medullary  Ray.  — Sec- 
tions should  be  cut  in  various  direc- 
tions and  at  different  levels  in  the 
cortex.  One  of  the  most  instructive 
is  to  cut  a section  across  the  direc- 
tion of  the  medullary  rays.  In  it 
will  be  seen  groups  of  transverse 
sections  of  the  various  tubes — col- 
lecting, ascending,  and  descending 
portions  of  the  looped  tubule  of 
Henle — which  make  up  a medul- 
lary ray.  Between  the  rays  there 
are  sections  of  glomeruli  and  con- 
voluted tubules. 

Blood-Vessels  of  the  Kidney. — The  sections  should  be  cut 
from  a kidney  injected  with  carmine  gelatine  or  Berlin-blue  gela- 
tine ; they  should  be  radial,  not  too  thin  ; best  from  the  kidney  of 
a small  mammal,  and  mounted  in  balsam.  In  injecting  a kidney 
do  not  use  too  great  pressure,  as  otherwise  the  glomerular  capill  tries 
are  apt  to  burst,  and  the  injection-mass  passes  into  the  tubules. 
When  the  stellate  veins  on  the  surface  are  seen  to  be  well  injected, 
one  may  infer  that  the  mass  has  traversed  the  glomerular  capillai  ies. 
In  a small  animal  inject  from  the  aorta,  in  a large  one  from  the 
renal  artery. 

4.  T.S.  Injected  Kidney. — (a.)  (L)  Between  the  cortex  and 
the  medulla,  i.e.,  in  the  boundary  or  intermediate  layer,  sections 
of  the  larger  branches  of  the  renal  artery  and  vein  will  be  seen 
(fig.  293,  RA),  ^.e.,  along  the  ‘Hine  of  vascular  supply.”  From 


ules ; C,  D,  Wide  and  narrow- 
parts  of  Henle’ s tubule ; E,  F. 
Blood-vessels. 


Medulla  Cortex. 


310 


PEACTICAL  HISTOL'OGY. 


[XXVIII. 


Fig.  299.— Blood-Vessels  of  the  Kidney. 
A Capillaries  of  cortex ; J^.  Of 

medulla;  a.  Interlobular  artery;  i. 
Vas  atferens  ; 2 Vas  efferens ; r,  e. 
Vasa  recta;  FF  Interlobular  vein  ; 
S.  Origin  of  a stellate  vein  ; i.  Bow- 
man’s capsule  and  glomerulus ; P. 
Apex  of  papilla ; C.  Capsule  of  kid- 
ney; e.  Vasa  recta  from  lowest  vas 
efferens. 


these  the  interlobular  arteries  and 
veins  (fig.  299,  a),  running  out- 
wards in  the  middle  between  every 
two  medullary  rays  (fig.  293). 
From  these  are  given  off  on  all 
sides,  at  short  intervals  along  the 
coarse  of  the  vessels,  short  arteries 
- — the  vasa  afferentia.  Each  vas 
afferens,  after  a very  short  course, 
runs  to  a Malpighian  capsule,  and 
splits  up  into  capillaries  to  form 
the  glomerulus. 

(6.)  If  the  capsule  is  cut  at  the 
level  where  the  artery  enters,  the 
short  and  narrower  efferent  vein  or 
vas  efferens  will  be  seen  coming 
out  at  the  same  pole,  and  after  a 
similar  short  course  breaking  into 
a network  of  capillaries,  which  sur- 
rounds the  tubules  of  the  cortex. 
Around  the  convoluted  tubules  the 
meshes  of  the  network  are  some- 
what polygonal,  but  in  the  medul- 
lary rays  in  the  cortex  they  are 
more  elongated. 

(c.)  From  this  capillary  network 
arise  short  veins,  which  join  the 
interlobular  veins  (fig.  299).  Quite 
at  the  upper  end  of  the  inter- 
lobular artery  a few  branches  are 
given  off,  which  end  directly  in 
this  capillary  network  without  the 
intervention  of  glomeruli.  Under 
the  capsule  the  small  veins  are 
arranged  in  a stellate  manner,  con- 
stituting the  vence  stellatce  (fig. 
299,  S).  ^ ^ 

{d.)  Passing  again  to  the  ‘‘line 
of  vascular  sup)>ly,’^  the  short 
vessels  which  break  up  Into  leashes 
or  bundles  of  small  blood-vessels 
(fig.  299,  r),  the  vasa  recta  (arteriae 
rectae.,  venae  rectae),  which  run  be- 
tween the  medullary  rays  into  the 
medulla,  where  they  form  an 
elongated  capillary  mesh  work  be- 


KIDNEY. 


XXVIII.J 


311 


tween  the  straight  tubules  of  the  medulla.  The  medulla  is  not  so 
vascular  as  the  cortex,  and  it  has  no  glomeruli. 

5.  Injected  and  Stained  T.S.  Kidney. — A not  too  thin  injected 
section — say  injected  with  a blue  mass — may  he  stained  with  picro- 
carmine,  which  makes  the  tubular  structures  more  distinct  by 
staining  their  nuclei. 

6.  Fresh  Kidney — Glomeruli  and  Basement  Membranes  of 
Tubules. — Expose  a fresh  kidney  to  the  air  for  a day  or  two 
according  to  the  temperature.  Cover  it  to  prevent  evaporation. 
Tease  part  of  the  medulla  and  cortex  in  normal  saline. 

(a.)  (H)  Observe  the  lon'g,  partly  empty,  structureless,  basement 
membranes  of  the  tubules,  often  exhibiting  folds ; 
also  isolated  cells  of  the  tubules. 

(b.)  Isolated  glomeruli,  consisting  of  several 
tufts  of  capillaries.  The  nuclei  of  their  cells  are 
revealed  by  dilute  acetic  acid. 

(c.)  Narrow  tubules,  not  unlike  blood-capih 
laries,  but  they  possess  a wall  lined  by  a layer  of 
squames,  the  nucleated  part  of  the  squames  alter- 
nating on  opposite  sides  of  the  tubule.  These  are 

the  descending  part  of  the  looped  tubule  (fig.  Fig.  300.— Part  of  De- 
\ sceiiding  Looped 

300^.  Tubule  of  Heiile. 

7.  Isolated  Cells  of  the  Different  Tubules. — 

Place  very  small  pieces  (size  of  a split-pea)  24-48  hours  in  a 5 per 
cent,  solution  of  neutral  ammonium  chromate.  Wash  in  water,  and 
tease  small  fragments  in  a 50  per  cent,  solution  of  potassic  acetate, 
or,  without  washing,  tease  a fragment  in  the 
chromate  solution. 

(a.)  (H)  Note  specially  the  cells  of  the  con- 
voluted tubules  and  those  of  the  ascending  limb 
of  Henle’s  loop.  They  show  the  “ rodded  char- 
acter of  the  outer  part  of  the  protoplasm.  Ad- 
joining cells  tend  to  interlock  with  each  other 
(fig.  301). 

8.  Isolated  Tubules. — Place  small  pieces  (size 
of  a pea)  of  the  kidney  of  a mouse,  tortoise,  or 
guinea-pig  (3-4  hours)  in  pure  hydrochloric  acid 
or  40  per  cent,  nitric  acid  (2-4  hours),  wash  in 
water  and  leave  them  in  water  for  18-24  hours. 

They  swell  up,  and  their  constituents  readily  fall 
asunder.  Place  a fragment  in  water  slightly  tinged  with  iodine  and 
gently  tap  the  glass  slide,  or  stain  with  dilute  acid-fuchsin.  This 
is  sufficient  to  cause  the  tubules  to  fall  asunder. 

(L  and  H)  In  a part  from  the  cortex  search  for  a convoluted 
tubule  still  connected  with  its  capsule,  the  twists  on  the  tube  itself, 


Fig.  3or.  — Isolated 
Cells  from  Convo 
luted  TubuleSc  i 
On  the  flat  with 
interlocking  pro- 
cesses; 2.  On  edge 
and  “ rodded.’’ 
Ammonium  chro 
mate. 


312 


PRACTICAL  HISTOLOGY. 


[XXVIII. 


and  the  transition  to  the  narrow  part  of  the  looped  tubule  of  Henle. 
This  is  perhaps  easiest  to  obtain  from  a mouse’s  kidney.  Isolated 
straight  tubules  from  the  medullary  part.  To  preserve  this  prepara- 
tion, suck  away  the  fluid  and  gently  replace  it  with  glycerine. 

Ureter  and  Bladder. — Harden  small  pieces  in  Muller’s  fluid 
(14  days)  or  in  corrosive  sublimate  (5-6  hours),  and  then  in 
gradually  increasing  strengths  of  alcohol.  Make  transverse  sections 
of  the  one  and  vertical  sections  of  the  other.  Flemming’s  fluid  for 
epithelium.  Stain  in  haematoxylin  and  mount  in  balsam ; and 
others  in  picro-carmine  and  mount  in  Tarrant’s  solution.  Or  stain  in 
bulk  in  borax-carmine  and  cut  in  paraffin.  The  ureter  of  a cat  or 
monkey  does  very  well,  and  it  is  well  to  use  the  contracted  bladder 
of  a small  mammal. 

9.  T.S,  Ureter  (fig.  302). — (a.)  (L)  Externally  is  a thin  fibrous 


"Adventitia. 


Fig.  302. — T.S.  Lower  Part  of  Human  Ureter,  g.  Transitional  epithelium  ; s.  Suh- 
miicosa ; I and  r.  L<  ngitudinal  and  circular  smootli  muscular  libies;  t.  Tunica 
propria.  Muller’s  fluid,  x 15. 


coat  or  adventitia,  consisting  of  connective  tissue  with  the  large 
vessels  and  nerveSc 

{b,)  The  muscular  coat  consists  of  an  outer  layer  of  smooth 
muscle  arranged  circularly^  and  inside  tins  a longitwHnal  coat 
arranged  in  bundles,  the  latter,  of  course,  divided  transversely.  In 
the  lower  part  of  the  ureter  there  is  an  incomplete  longitudinal 
muscular  coat  outside  the  circular  coat. 

[r,)  The  submucous  coat  is  thin,  and  passes  gradually  into 
{d.)  The  mucous  coat,  which  is  thrown  into  ridges  or  folds,  and 
is  lined  by  transitional  epithelium. 


XXVIII.] 


KIDNEY. 


3'3 


Fig.  303.—  V.S.  Epithelium  of  the  Mucous  Membrane 
of  a Human  Bladder.  Muller’s  tiuid,  x 560. 


(e.)  (H)  The  transitional  epithelium,  and  the  variation  in  the 
shape  of  the  cells  arranged  in  several  layers,  from  the  free  mucous 
surface  outwards. 


10.  V.S.  Bladder  (fig. 

303). — (a.)  (L)  Most  ex- 
ternally a thin  fibrous  coat, 
in  some  places  covered  by 
a serous  coat. 

(/;.)  The  muscular  coat, 
composed  of  longitudinal 
and  circular  smooth  fibres. 

Usually  there  is  an  outer 
and  an  inner  longitudinal 
layer  with  a circular  layer 
between.  The  appearance 
of  these  layers  will  depend 
on  the  plane  of  the  sec- 
tion. 

The  submucous  and  mucous  (H)  coats  are  like  those  of  the 
ureter. 

(c.)  (H)  The  transitional  epithelium  and  the  great  variety  in  the 
shape  of  the  cells  from  below 
upwards.  Occasionally  amongst 
the  epithelial  cells  are  leucocytes 
(fig.  303)0  It  is  important  to 
observe  that  the  thickness  and 
shape  of  the  lining  transitional 
epithelium  will  necessarily  vary 
with  the  state  of  distension  or 
contraction  of  the  bladder. 

11.  T.S.  Penis,  e.g.,  of  a 

monkey  or  other  small  mammal. 

Harden  it  in  alcohol  or  fix  in 
Flemming’s  fluid.  Make  T.S. 
and  stain  with  safranin. 

(a.)  (L)  It  consists  of  the  two 
corpora  cavernosa,  placed  dor- 
sally,  one  on  each  side  of  the 

middle  line,  and  interiorly  the  304  —t.s.  Penis  of  Monkey.  CC.  Cor- 
’ pus  cavern osum  ; CS.  Corpus  spongio- 

corpus  spongiosum.  In  the  sum;  s.  Septum;  u.  Urethra:  DV, 
centre  of  the  latter  is  the  vein ; Dorsal  arteries ; ^ 

urethra  as  a transverse  slit. 

In  the  prostatic  part  the  mucous  membrane  is  lined  by  transitional 
epithelium,  but  in  the  body  of  the  penis  it  is  lined  by  the  columnar 
variety,  except  at  the  meatus,  where  it  is  stratified. 

28 


PRACTICAL  HISTOLOGY. 


314 


[XXVIII 


(6.)  N^ote  the  trabeculae  of  connective  tissue — bounding  wide 
spaces — in  the  cavernous  part.  The  whole  is  surrounded  by  a 
tough  capsule,  in  which,  dorsally,  are  sections  of  blood-vessels — one 
vein  (DV)  and  two  arteries  (DA) — and  nerves  (fig.  304,  N). 

/ SUPEARENAL  CAPSULE. 


It  is  well  to  remember  that  there  are  great  variations  in  the 
structure  of  this  gland  in  different  animals.  The  suprarenal 
capsule  is  a ductless  gland,  consisting  of  a cortical  zone  and  a 


Zona  glomerulosa. 


Z.  fasciculata. 


Z.  reticularis. 


Strands  of  cells  of 
the  medulla. 


T.S.  of  a nerve. 


Ganglionic  cells. 

T.S.  bundles  of 
smooth  muscle. 


T.S.  vein. 


Capsule. 


Cortex. 


Medulla. 


Fig.  305.— T.S.  Human  Suprarenal  Capsule,  x 50. 


medulla.  It  is  invested  by  a fibrous  capsule  which  sends  septa 
into  the  gland.  Especially  in  the  cortex,  these  septa  run  so  as  to 
give  a columnar  arrangement  to  the  cells  which  lie  between  them. 
The  parenchyma  of  the  organ  consists  of  cells  which  vary  in  their 
characters  in  different  regions.  The  cells  of  the  cortex  (15  are 
polyhedral,  nucleated,  granular,  yellowish-coloured  cells,  arranged 
under  the  capsule  in  rounded  grou[)s — zona  glomerulosa.  !N^ext 
this  is  the  widest  zone — the  zona  fasciculata.  Next  the  medulla 


XXVIII.] 


SUPRARENAL  CAPSULE. 


315 


is  the  zona  reticularis.  In  the  medulla  the  cells  are  often  irregular 
or  polygonal  with  a clearer  protoplasm,  which  is  often  tinged  of  a 
yellowish  or  brownish  colour.  There  are  numerous  vessels  and 
nerves,  the  latter  with  ganglionic  cells. 

Harden  the  suprarenal  capsules  of  a guinea-pig  in  Klein’s  fluid 
(5-7  days),  and  then  in  alcohol,  or  fix  in  Flemming’s  fluid.  Harden 
a human  suprarenal  in  Kleinenberg’s  fluid  (24  hours),  and  then  in 
alcohol.  Make  radial  sections,  and  stain  some  in  hsematoxylin,  and 
others  in  picro-carmine.  Or  stain  and  cut  in  paraffin. 

12.  V.S.  Suprarenal  Capsule. — (L)  Observe  the  arrangement 
already  described.  It  is  to  be  noted,  however,  that  there  are  great 
variations  in  the  structure  of  these  organs  in  different  species  of 
animals, 

(|-j)  Examine  the  cells  in  the  various  zones  (fig.  305). 


ADDITIONAL  EXERCISE. 

Termination  of  Nerves  in  Suprarenal  Capsules. — The  literature  and  most 
recent  results  will  be  found  iii  Fusari’s  paper^  (with  a ]»late).  He  used 
capsules  of  the  mouse,  rabbit,  pi^,  cat,  and  new-born  infant.  The  method 
employed  was  the  quick  method  of  GoLi  (Lesson  XXX.),  i.e.^  small  fresh  pieces 
are  placed  in  the  osmn^o-bichromate  fluid  (3-10  days),  and  afterwards  in  i per 
cent,  silver  nitrate  solution  (1-2  days). 


LESSON  XXIX. 

SKIN  AND  EPIDERMAL  APPENDAGES. 

THE  SKIN. 

The  skin  consists  of  the  epidermis  and  cutis  vera,  dermis,  or 
corium.  The  epidermis  consists  of  many  layers  of  stratified 
squamous  epithelium  (p.  317).  The  corium  is  composed  of  a basis 
of  fibrous  connective  tissue — white  and  yellow  fibres— and  its  sur- 
face is  thrown  into  a number  of  papillae,  which  differ  in  size, 
number,  arrangement,  and  form  in  different  parts  of  the  body. 
Undivided  conical  elevations  are  called  simple  but  when 

these  are  beset  with  smaller  papillae,  they  are  called  compound 
papillcB,  The  epidermis  completely  covers  in  the  apices  of  the 

^ Archiv  ited.  de  Biol.y  xvi.  p.  262,  1891. 


3i6 


PRACTICAL  HISTOLOGY. 


[xxix. 


papillae,  and  also  dips  down  into  the  furrows  between  adjoining 
rows  of  papillae,  so  that  the  surface  of  the  skin  is  smooth,  although 
the  arrangement  of  the  papillae  is  readily  detected  by  the  lines  on 
the  palmar  aspect  of  the  hand  and  foot.  The  fibrous  tissue  of  the 
cutis,  next  the  epidermis,  forms  a very  thin  modified  layer  with 
scarcely  any  fibrils  and  no  corpuscles.  This  layer  acts  the  part  of 
a basement  membrane,  and  is  continuous  with  the  basement 
rhembrane  of  a sweat-gland.  In  the  dermis,  the  bundles  of  white 
fibres  interweave  with  each  other,  and  form  a dense  tissue ; at  the 
lower  part  of  the  skin  it  becomes  more  open  in  texture,  and  gradu- 
ally passes  into  the  subcutaneous  tissue.  Elastic  fibres  in  the  form 
of  networks  exist  in  large  numbers  in  the  cutis ; they  are  finer  in 
the  papillae,  and  coarser  lower  down. 

The  subcutaneous  tissue  consists  of  a complex  system  of 
trabeculae  of  fibrous  tissue,  and  in  some  of  the  meshes  are  lobules 
of  fatty  tissue  forming  a fatty  layer,  constituting  the  stratum 
adiposum. 

The  arrangement  of  the  hlood-vessels  is  stated  at  p.  325.  There 
are  also  numerous  lymphatics  and  nerves — some  of  the  latter  with 
peculiar  terminations — glands  (sweat  and  sebaceous),  and,  in  some 
situations,  hairs  with  their  hair-follicles. 

It  is  important  to  distinguish  between  the  hairy  skin  and  the 
parts  of  the  skin  without  hairs.  The  non-hairy  parts  are  the  volar 
surfaces  of  the  hands,  feet,  fingers,  and  toes,  nails,  lips,  mammary 
papillae,  certain  parts  of  the  external  genitals,  and  the  inner  part  of 
the  external  auditory  meatus.  The  hairy  parts  are  the  remainder 
of  the  skin.  The  non-hairy  parts  are  concerned  with  direct  tactile 
sensations,  the  hairy  parts  with  indirect  tactile  sensations,  the  hairs 
themselves  being  the  chief  tactile  organs  {Blaschko'^),  This 
observer  has  shown  that  the  epidermis  projects  into  the  cutis  vera 
in  the  form  of  septa,  varying  in  form  and  distribution  in  different 
parts  of  the  skin  (p.  325). 

Methods. — The  skin  must  be  prepared  in  various  ways  according 
to  the  particular  part  which  it  is  desired  to  study.  For  a general 
view  proceed  as  follow  : — (a.)  Procure  a fresh  portion  of  human 
skin  from  the  palm  of  the  hand  or  sole  of  the  foot,  cut  it  into 
pieces  about  i cm.  square,  and  remove  most  of  the  subcutaneous  fat ; 
pin  it,  epithelial  surface  downwards,  on  a piece  of  cork,  and  harden 
it  in  absolute  alcohol  (12  hours).  Renew  the  alcohol  for  another 
twenty-four  hours.  Sections  may  be  cut  by  freezing,  and  then 
stained  with  haematoxylin  or  picro-carmine  (the  latter  to  be  mounted 
in  FarranPs  solution).  Better  still,  stain  the  whole  ‘‘in  bulk”  in 
borax- carmine  or  haematein,  and  embed  and  cut  it  in  paraffin. 


^ Arcliiv  f.  mik.  Anat.^  xxx.  p.  495. 


THE  SKIN. 


XXIX.] 


317 


Mount  in  balsam.  Or  skin  so  hardened  may  be  double  stained  in 
bulk,  first  in  borax-carmine  and  then  in  hsematein. 

(6.)  Harden  the  skin  in  Muller’s  fluid. 

(c.)  For  the  layers  of  the  epidermis  fix  say  small  pieces  of  the 
skin  in  i per  cent,  osniic  acid  or  Flemming’s  fluid,  and  harden  in 
alcohol.  Stain  sections  in  safranin. 

1.  V.S.  Skin,  Palm  of  Hand. 

{a.)  (L)  The  epidermis,  consisting  of.  many  layers  of  stratified 
squamous  epithelium,  resting  on  the  cutis  vera,  dermis,  corium,  or 
true  skin.  The  latter  consists  of  connective  tissue,  and  is  provided 
with  finger-shaped  elevations  or  papillae,  which  project  into  the 
deeper  layers  of  the  epidermis,  the  latter  in  the  form  of  septa,  dip- 
ping in  between  the  papillae  (fig. 

306) . 

(h.)  The  epidermis,  composed 
entirely  of  stratified  epithelial  cells. 

Proceeding  from  the  outside  (fig. 

307) ,  observe — ■ 

(i.)  The  stratum  comeum,  of 
variable  thickness,  consisting  of 
many  layers  of  flattened  or  slightly 
fusiform,  clear,  non -nucleated  cells 
united  to  each  other.  As  the  cells 
are  seen  on  edge,  they  are  very  thin. 

Those  on  the  surface  are  about  to 
be  shed,  and  consist  of  keratin. 

(ii.)  The  stratum  lucidum,  a 
thin,  narrow,  clear,  homogeneous 
layer,  composed  of  two  or  more 
layers  of  flattened  cells,  containing 
sometimes  a rod  - shaped  nucleus. 

The  cells  do  not  stain  well  with 
dyes.  The  eleidin  granules  seeni  to 
become  fused  together  and  form  the  basis  for  cornification,  as  the 
cells  are  changed  and  become  corneous. 

(hi.)  The  stratum  granuiosum,  a somewhat  thicker  layer,  com- 
posed of  ovoid  cells  two  or  three  rows  deep.  Each  cell  is  distinctly 
granular,  and  usually  this  layer  stands  out  deeply  stained,  because 
its  granules  of  eleidin  or  keratohyalin  are  stained  whth  the  car- 
mine. The  cells,  like  the  foregoing,  are  devoid  of  “prickles.’' 

(iv.  ) Tl  le  stratum  Malpighii,  several  layers  of  more  plastic  cells. 
At  the  lowest  part,  w’here  they  rest  on  the  papillae  of  the  true  skin, 
the  cells — prickle-cells — are  smaller  and  columnar  in  shape  (with 
oval,  vertically-placed  nuclei),  but  above  this  they  become  more 
spheroidal  or  polygonal,  and  each  one  is  distinctly  nucleated. 


Fig.  306. — v s.  Skin  of  Palmar  Surface 
of  Finger.  F.  Fat ; P.  1\S.  Pacinian 
corpuscle. 


3i8 


PKACTICAL  HISTOLOGY. 


[xxix. 


(c.)  The  cutis  vera.  The  papillae,  conical  elevations  projecting 
into  the  Malpighian  layer.  They  consist  of  compact  fibrous  tissue. 
The  rest  of  the  skin  consists  of  bundles  of  white  fibrous  tissue 
interwoven  with  networks  of  elastic  fibres,  and  at  its  lower  part 
masses  of  fat-cells.  The  connective  tissue  and  fat-cells  below 
become  continuous  with  the  subcutaneous  tissue,  which  is  of  a 
more  open  texture ; but  there  is  a gradual  transition  from  the  one 

to  the  other.  The  nuclei  of  the 
connective  tissue  corpuscles  appear 
as  red  oval  dots. 

In  sections  of  the  sudoriferous 
glands,  their  coils  (in  the  deeper 
layers  of  the  corium),  their  ducts 
running  vertically  through  the 
skin,  and  a corkscrew  passage  in 
the  epidermis  may  be  seen.  In 
some  of  the'  papillae  observe  a 
touch-corpuscle  (p.  320),  and  in 
the  subcutaneous  tissue  occasion- 
ally sections  of  Pacinian  bodies 
(p.  320). 

, i'f-)  (H)  Observe  in  the  epi- 
dermis the  shape  and  characters 
of  the  successive  layers  of  epi- 
thelium. In  the  Malpighian 
layers,  “prickle-cells,”  f.e.,  cells 
connected  with  each  other  by 
fine  “inter-cellular  bridges,”  are 
better  studied  in  an  osmic  acid 
section  (fig.  97).  (See  also 
Lesson  IV.) 

(i.)  In  the  stratum  Malpighii 
the  lowest  cells  are  arranged  in 
a single  layer  of  elongated,  some- 
what columnar  cells  (6-12  /x,),’ 
with  large  oval  nuclei  surrounded 
by  granular  protoplasm.  The 
lower  ends  of  the  cells  frequently  exhibit  processes  which  fit  into 
the  dermis.  The  remainder  of  the  cells  of  this  layer  are  irregularly 
cubical,  and  exhibit  prickles  (Lesson  IV.,  and  p.  127).  In  the  dark 
races  the  particles  of  melanin,  which  give  the  dark  colour  to  the 
skin,  are  present  in  the  cells  of  this  layer,  especially  in  the  deepest 
layer  of  cells.  Nuclei  are  sometimes  seen  in  process  of  division. 

(ii.)  The  cells  of  the  stratum  granulosum  are  arranged  in  two  or 
more  layers,  and  are  flattened  horizontally,  so  that  they  are  lozenge- 


n 

Fia.  307. — V.S.  Hnman  Fp  dermis  with 
Terminations  of  JVerve  - Fibrils,  n. 
Kerve ; d.  Dermis ; &.  Branches  of 
nerve-fibrils. 


XXIX.] 


THE  SKIN. 


.319 


shaped,  devoid  of  prickles,  and  are  crowded  with  granules  of  eleidin. 
a substance,  apparently  one  of  the  stages  on  the  way  to  the  body 
keratin.  These  cells  stain  deeply  with  picro-carmine,  hseinatoxylin, 
and  osmic  acid.  They  are  soluble  in  caustic  potash,  and  in  this 
respect  differ  from  keratin. 

(iii.)  The  stratum  lucidum  is  composed  of  two  or  more  layers  of 
flattened  transparent  cells,  with  no  prickles,  and  free  from  granules, 
but  with  a horizontally-placed,  rod-shaped  nucleus.  They  do  not 
stain  readily. 

(iv.)  The  stratum  corneum  or  horny  layer  consists  of  horny 
squames  composed  of  keratin  (Lesson  IV^). 

(e.)  The  sweat-glands  are  most  numerous  in  the  palm  of  the 
hand  and  sole  of  the  foot.  Each  gland  is  a simple  tube  coiled  up 
at  its  lower  extremity  into  a coil  inch  in  diameter.  To  see  their 
whole  course — coil  and  duct — the  sections  must  not  be  too  thin, 
and  should  be  parallel  to  the  course  of  the  gland.  The  coil  of  the 
gland  lies  in  the  subcutaneous  tissue.  The  secretory  part  of  the 
tube  consists  of  a basement  membrane  lined  by  a single  layer  of 
nucleated  transparent  cubical  or  columnar  cells  surrounding  a small 
but  distinct  lumen.  Between  the  epithelium  and  the  basement 
membrane  is  a longitudinally- 
disposed  layer  of  smooth 
muscular  fibres.  The  coil 
also  contains  a part  of  the 
sudoriferous  canal  or  duct 
(fig.  308).  The  latter  is 
narrower  than  the  secretory 
part,  and  consists  of  a base- 
ment membrane  lined  by 
several  layers  of  polyhedral 
cells.  There  is  no  muscular 
layer,  but  internal  to  the 
epithelial  lining  there  is  a 
delicate  membrane  or  cuticle. 

Trace  the  coil  into  its  duct,  which  runs  vertically  through  the  cutis 
vera  with  a slightly  wavy  course.  It  has  a basement  membrane 
lined  by  two  or  three  layers  of  short  cubical  cells,  which,  if  traced 
upwards,  become  continuous  with  those  of  the  ^lalpighian  layer  of 
the  epidermis,  looking  like  a funnel-shaped  expansion.  The  lumen 
of  the  duct  is  distinct.  The  basement  membrane  becomes  continuous 
with  the  altered  superficial  layer  of  the  corium  just  under  the  epi- 
dermis. The  lumen  of  the  duct  is  continued  upwards  in  a corkscrew 
spiral  through  the  epidermis.  A complete  view  of  its  course  is  only 
obtained  in  a thickish  section.  In  a thin  section  the  twistings  are 
of  course  divided. 


Fig.  308.— Section  of  Part  of  Coil  of  a Sweat- 
Gland.  D.  Duct;  S.  Secretory  part,  x 300. 


320 


PRACTICAL.  HISTOLOGY. 


[xxix. 

2.  V.S.  Skin  of  Negro  (H). — Harden  in  alcohol,  and  stain  the 
sections  slightly  in  eosin.  Mount  in  balsam.  Observe  the  granules 
of  melanin  in  the  deepest  layers  of  the  epidermis. 

3.  V.S.  Skin  of  Finger  {Double  Stained).— a section  first 
in  methyl-green  iodide  and  clarify  it  with  clove-oil  coloured  with 
eosin.  Wash  out  the  clove-oil  with  xylol  and  mount  in  balsam. 
The  stratum  corneum  is  green,  and  so  are  the  nuclei  of  the  other 
epidermic  and  connective-tissue  cells. 

4.  Prickle-Cells  and  Touch-Corpuscles.— Place  in  i per  cent, 
osniic  acid  or  Flemming’s  fluid  (24  hours)  a very  small  piece  of 
fresh  skin  from  the  palmar  surface  of  a finger.  Wash  it  well  in 
water  and  complete  the  hardening  in  alcohol.  Make  V.S.  and 
mount  in  Tarrant’s  solution.  Or  cut  in  paraffin,  stain  in  safranin, 
and  mount  in  balsam. 

(a.)  (H)  Observe  the  various  layers  of  the  epidermis,  hut  in  the 
Malpighian  layer  note  the  prickle-cells.  The  cells  appear  to  bo 
joined  by  their  edges  by  fine  striae,  the  striae  - leaving  small  spaces 
between  them  (fig.  97).  The  striae  are  fine  “ intercellular  bridges,” 
stretching  from  one  cell  to  another,  and  it  is  only  when  the  epi- 
dermis is  dissociated  and  the  bridges  broken  that  these  cells  appear 
as  cells  beset  with  fine  spines,  and  hence  they  were  called  prickle- 
cells.” 

(h.)  In  a papilla  search  for  a Wagner’s  touch-corpuscle  (fig.  351). 
It  is  an  oval  body,  with  its  long  axis  in  the  long  axis  of  the  papilla, 
but  they  are  not  present  in  all  papillae  (fig.  350).  They  consist  of 
a fibrous-looking  material,  with  flattened  nuclei  arranged  trans- 
versely. To  their  lower  end  passes  a medullated  nerve-fibre,  which 
usually  twists  round  the  corpuscle  before  it  enters  it.  The  ultimate 
distribution  of  the  nerve  is  best  seen  in  a gold  chloride  preparation 
(Lesson  XXXIV.). 

5.  V.S.  Foetal  Skin  for  Sweat-Glands  and  Pacinian  Corpuscles. 

— Harden  the  skin  of  the  tips  of  the  fingers  of  an  infant  in  alcohol 
and  make  V.S.  Stain  with  hsematoxyiin  and  eosin. 

{a.)  (L)  Observe  the  general  arrangement  already  described,  but 
the  sweat-glands  are  much  more  closely  placed  than  in  adult  skin, 
and  there  is  less  intervening  connective  tissue.  A child  at  birth 
has  its  full  complement  of  sweat-glands,  and  hence  they  must  be 
more  crowded  together  than  in  the  adult. 

{h.)  In  the  subcutaneous  tissue  are  lobules  of  fat  and  sections  of 
Pacinian  corpuscles  (Lesson  XXXIV.  5).  The  latter  appear  to  con- 
sist of  concentric  laminae  surrounding  a central  core. 

{c.)  (H)  Observe  two  or  three  layers  of  more  or  less  cubical  cells 
lining  the  duct  of  the  gland,  while  the  true  secretory  portion  is 
lined  by  a single  layer  only  of  low,  clear,  columnar  cells. 

Hair  Follicles. — To  see  their  structure,  harden  the  scalp  in 


THE  SKIN. 


321 


XXIX.J 

alcohol.  V.S.  must  be  made  parallel  to  the  course  of  the  hair- 
follicles,  which  requires  some  care.  Others  are  made  across  the 
follicles  at  different  levels ; hut  in  this  case  care  must  be  taken  not 
to  make  the  section  parallel  to  the  surface  of  the  skin,  hut  at  right 
angles  to  the  course  of  the  hair-follicle.  If  an  oblique  section  be 
cut,  the  hair-follicles  are  cut  at  different  levels. 

Harden  a small  piece  of  the  human  scalp  (2  cm.,  square)  in 
Muller’s  fluid  and  afterwards  in  alcohol.  Stain  a section  in  log- 
wood, or  first  with  logwood  and  then  with  picro-carmine.  Mount 
in  balsam. 

6.  V.S.  Hair-Follicle. — {cl)  (L)  and  (H)  Observe  the  very  thin 
epidermis,  the  thick  cutis  vera,  and  deep  down  the  subcutaneous 
masses  of  fat;  the  hair-follicles,  running  obliquely  through  the 
skin,  each  one  with  a la!r  in  it.  At  the  lower  ))art  the  hair  has  a 
bulbous  end  implanted  on  a papilla ; the  various  coats  of  the  hair- 
follicle,  some  continuous  with  the  corium,  and  others  with  the 
epidermis.  The  following  scheme  shows  the  layers  of  the  hair- 
follicle 


Coverings  of  a Hair-Follicle  from  Without  Inwards. 


I.  Fibrous  layers 


’ {a. ) Longitudinally-arranged  fibrous  tissue. 
{b. ) Circularly-arranged  spindle-cells. 


2.  Glass-like  or  hyaline  membrane. 


!(a.)  Outer  root-sheath,  f Heule’s  layer. 

(ft.)  Inner  root-sheath.  1 Huxley’s  layer. 

(c.)  Cuticle  of  the  hair.  (Cuticle  of  root-sheath. 


4.  The  hair. 


(h.)  Dermic  Coverings. — (i.)  {a.}  The  outer  fibrous  sheath 
denser  than  and  continuous  with  the  corium.  The  fibres  run  for 
the  most  part  longitudinally,  (b.)  The  inner  fibrous  sheath  of 
fibrous  tissue  has  a more  circular  arrangement  and  is  seen  as  fibres 
cut  across  transversely,  with  a few  nuclei  interspersed. 

(2.)  The  hyaline  or  basement  membrane,  clear,  structureless,  and 
well  marked.  It  separates  the  dermic  from  the  epidermic  coverings 
of  the  hair. 

(3.)  The  Epidermic  Coverings.— (u.)  The  outer  root-sheath— 
most  obvious  part  of  the  covering — consisting  of  several  layers  of 
nucleated  cubical  cells,  continuous  with  and  resembling  those  of  the 
Malpighian  layer. 

{b.)  The  inner  root-sheath,  much  narrower  and  paler,  consisting 
of  three  layers  of  cells  of  different  characters,  is  present  only  in  the 
lower  part  of  the  follicle,  i.e.,  below  the  sebaceous  gland. 

(4.)  The  hair  with  its  cuticle. 

{a.)  At  its  lowest  part  the  bulbous  enlargement  of  the  hair,  with 
29  X 


322 


PRACTICAL  HISTOLOGY. 


[XXIX. 


the  papilla  of  the  hair  follicle  (fig.  309)  projecting  into  it,  and  con- 
tinuous with  the  corium. 

{h.)  The  sebaceous  gland  or  glands.  In  a balsam  preparation 


Fig.  30Q.— V.S.  Hair-Follicle  of  Human  Scalp.  i and  2.  Outer  and  inner  fibrous  sheaths 
of  hair-follicle ; 3.  Hyaline  layer ; 4.  Outer,  and  5 and  6.  Inner  root-sheaths ; p.  Root 
of  hair,  with  its  papilla;  A.  Arrector  pili  muscle;  C.  Cutis  vera;  a.  Subcutaneous 
tissue,  with  fat  lobules ; h.  Epidermis  (horny  layer) ; d,  Rete  Malpighii ; g.  Blood- 
vessels ; V.  Lymphatics  of  papilla ; h.  Fibrous  part,  i.  Medulla,  k.  Cuticle  of  hair ; 
K,  Sweat-gland  and  its  coil. 


its  acini  are  yellowish  and  clear,  opening  by  a duct  into  the  hair- 
follicle  at  about  its  upper  third  (fig.  309,  'J'). 

(c.)  The  arrector  pili  muscle  (smooth  muscle)  stretching  ob- 


XXIX.]  THE  SKIN.  323 

liquely  from  the  deeper  part  of  the  hair-follicle  to  the  upper  part  of 
the  corium  (fig.  309). 

7.  T.S.  of  Hair-Follicles  (Scalp). 

{a,)  (L)  In  the  human  scalp  the  hair-follicles  are  arranged  in 
groups  of  three  or  four,  with  interweaving  strands  of  connective 
tissue  between  them.  The  various  coverings — dermic  and  epi- 
dermic— can  now  be  distinctly  seen,  especially  if  the  section  be 
through  the  lower  half  of  the  hair-follicle. 

(6.)  (H)  Observe  both  V.S.  and  T.S.  to  see  the  structural  ele- 
ments forming  the  outer  coverings  of  a hair-follicle. 

(i.)  The  inner  rooi-sheath  consists  of  an  outer  layer  of  cells,  clear 
and  non-nucleated — Henle^s  layer — and  an  inner  nucleated  layer — 
Huxley's  layer.  Both  are  best  seen  in  T.S. 

(c.)  The  hair  has  a cuticle^  while  the  hair  itself  may  or  may  not 
have  a medulla. 

Carefully  compare  the  structures  of  the  hair-follicle  in  the  T.  and 
V.  sections. 

{d.)  The  sebaceous  gland,  its  acini  lined  by  cubical  cells,  contain- 
ing fat,  and  rendered  clear  by  the  balsam. 

The  epithelium  of  the  duct  continuous  with  that  of  the  outer 
root-sheath. 

8.  Sebaceous  Glands. — (a.)  Harden  the  alae  of  the  nose  of  a 

new-born  child  in  corrosive  sublimate,  {h.)  Or  the  ala  of  nose  or 
adult  scrotum  in  picro-sulphuric  acid  (2-3  hours).  Stain  all 
with  haematoxylin.  Large  sebaceous  glands  opening  free  on  the 
surface  without  any  hair-follicle  are  found.  In  other  situations 

they  open  into  the  neck  of  a hair  follicle.  They  are  saccular 

glands  with  oval  alveoli,  which  lead  into  a short  duct.  The  alveoli 
are  lined  by  a layer  of  polyhedral  cells,  and  internal  to  this  are 
larger  cells  containing  fatty  matter.  The  sebaceous  secretion  is 
formed  by  these  cells  undergoing  disintegration,  and  liberating  the 
fatty  matter  they  have  formed.  They  are  developed  from  the  outer 
root-sheath.  In  balsam  they  are  clear,  but  in  water  they  appear 
dark  and  granular. 

9.  Human  Hair  (H). — Place  it  in  water,  cover,  and  examine. 

A rod-shaped  body  covered  by  a single  layer  of  thin,  non-nucleated 
transparent  imbricate  scales  arranged  transversely — cuticle.  In 

some  hairs  it  is  seen  merely  as  fine,  more  or  less  transverse, 
irregular,  or  wavy  lines  joining  each  other.  These  indicate  where 
the  one  cell  overlaps  the  other.  The  substance  or  cortex  of  the 
hair,  composed  of  horny,  fibrous  substance — hair  fibres — finely 
striated  longitudinally,  with,  in  some  hairs,  fine  pigment-granules 
scattered  along  the  course  of  the  hair  between  the  hair-fibres.  In 
some  hairs  a darker  central  core  or  wxdulla  composed  of  polyhedral 
cells. 


324 


PRACTICAL  HISTOLOGY. 


[XXIX. 


10.  Elements  of  a Hair  (H). — Place  a small  piece  of  a hair  in 
a drop  of  strong  sulphuric  acid.  Cover  and  press  lightly  with  a 
needle.  Be  careful  to  avoid  letting  a drop  of  the  acid  fall  on  the 
hrasswork  of  the  microscope.  The  hair  splits  up  longitudinally 
into  what  look  like  fibres,  but  by  gentle  tapping  on  the  cover  they 
split  into  cells,  so  that  a hair  is  composed  of  epithelial  cells  joined 
together,  having  previously  undergone  conversion  into  keratin. 

11.  Babbit’s  Hair  (H).  — Mount  in  balsam.  This  hair  contains 
one  or  more  rows  of  cubical  cavities  containing  air.  The  cavities 
appear  black,  and  are  surrounded  by  a small  quantity  of  cortex. 

12.  Wool  (Lesson  I.  10). 


Huxley’s  layer) : h.  Cuticle  ; 1.  Surface  of  Finger. 

Hair. 

Blood-Vessels  of  the  Skin. — Y.S.  of  a piece  of  skin  cut  from  a 
limb  injected  with  a gelatine  mass.  They  must  not  be  too  thin. 
After  injection  the  skin  is  hardened  in  Muller’s  fluid  and  afterwards 
in  alcohol  (three  weeks).  Mount  (balsam)  a section  of  the  palmar 
surface  of  a finger,  and  one  from  the  general  surface  of  the  body. 

A good  injection-mass  is  a watery  solution  of  china-ink.  It  is 
rubbed  down  on  a hone  until  a moderately  thick  black  solution  is 
obtained,  so.  that  when  a drop  is  placed  on  blotting-paper  it  holds 
together,  and  no  grey  ring  is  formed  round  the  drop.  It  has  this 


XXIX.]  THE  SKIN.  325 

advantage,  that  it  is  not  changed  by  exposure  to  light,  but  the 
tissue  must  be  hardened  before  sections  are  cut. 

13.  V.S.  Injected  Skin,  e,g.^  Palm. — The  section  should  be  cut 
in  paraffin,  and  include  the  subcutaneous  tissue  (fig.  31 1). 

{a.)  (L)  The  arteries  of  the  skin  are  branches  of  the  larger 
arteries  in  the  subcutaneous  tissue.  A branch  may  be  seen  running 
towards  the  surface.  In  its  course  it  gives  off  three  independent 
sets  of  branches,  which  end  in  capillaries  : — 

(i.)  The  lowest  to  the  groups  of  fat-cells,  where  it  forms  a net- 
work of  capillaries  around  and  between  the  fat-cells. 

(ii.)  The  short  branch  to  the  coil  of  a sweat-gland,  forming  a rich 
network  of  capillaries  between  the  coils  of  the  tube. 

(iii.)  The  highest  is  from  the  terminal  branches  of  the  artery, 
and  splits  up  into  capillaries,  which  form  a network  chiefly  in  the 
upper  part  of  the  corium,  and  from  this  branches  pass  which  form 
capillary  loops  in  the  papillae  of  the  skin.  From  it  also  proceed 
branches  to  the  hair-follicle  and  its  sebaceous  gland. 

{h.)  The  vein  arises  from  the  capillaries  of  the  papillae  and  the 
branches  of  the  arteries  to  the  upper  part  of  the  cutis,  and  in  its 
course — running  near  the  corresponding  artery — it  collects  the 
veinlets  from  the  coil  and  masses  of  fat.  For  blood-vessels  of  skin 
see  W.  Spatleholz.^ 

14.  Under-Surface  of  Epidermis. — Separate  by  one  of  the  follow- 
ing methods  the  epidermis  from  the  cutis,  most  easily  done  in  the 
foetus  : — (a.)  Use  a foetus  that  has  died  and  been  macerated  in  utero. 

(b.)  Place  pieces  of  skin  in  per  cent,  acetic  acid  (1-3  days), 
adding  a drop  or  two  of  chloroform  to  prevent  putrefaction 
(Philippson). 

(r.)  Macerate  at  40°  C. — preferably  foetal  or  young — skin  pinned 
out  on  cork  in  6 per  cent,  (or  weaker)  wood  vinegar  for  1-2  days. 
The  epidermis  separates  rapidly  (Loeivy).^ 

After  the  epidermis  peels  off,  in  all  cases  turn  its  deeper  surface 
upwards,  and  stain  it  for  3-4  minutes  with  a watery  solution  of 
BoehmeFs  logwood  (p.  68).  Wash  and  mount  in  balsam,  pre- 
ferably without  a cover-glass. 

• (L)  Observe  a system  of  septa  crossing  each  other,  and  forming 

longitudinal  and  transverse  ridges,  which  project  into  the  cutis. 
They  form,  as  it  were,  the  negative  picture ; the  papillae  of  the 
cutis  vera  represent  the  positive  image.  Part  of  the  cells  lining 
the  sweat-glands  and  hair-follicles  may  also  be  pulled  out,  and  are 
turned  toward  the  observer.  Figures  of  the  arrangement  of  these 
septa  are  given  in  the  papers  of  Blaschko  and  Loewy  (pp.  316,  325) 

^ Archiv  f.  Anat.  v.  Phys.,  Anat.  Abth.,  1893. 

^ Archivf,  mik,  Anat.,  xxxvii.  p.  159,  1891. 


326 


PRACTICAL  HISTOLOGY. 


[XXIX. 


THE  NAILS. 


The  body  of  the  nail  rests  on  the  nail-bed^  the  root  of  the  nail 
on  the  matrix,  and  the  part  at  the  root  and  sides  from  which  the 
nail  springs  is  the  nail-(jroove.  The  body  of  the  nail  is  made  np  of 
numerous  clear  horny  cells,  each  containing  a rod-shaped  nucleus. 
The  nails  are  really  the  stratum  lucidum,  the  stratum  corneum 
being  absent,  and  this  rests  on  the  Malpighian  layer  like  that  of 

the  epidermis.  The 
^ 4T  \ corium  or  nail-bed,  on 
which  the  nail  rests, 
is  beset  with  very 
vascular  longitudinal 
ridges^  papillae  being 
absent. 

Harden  the  nail  of 
a child  and  its  sub- 
jacent bed  in  alcohol. 
Make  T.  and  L.  sec- 
tions. Stain  in  hae- 
matoxylin  (balsam)  or 
picro  - carmine  (Tar- 
rant’s solution). 

15.  T.S.  Nail  (L 
and  H). — Observe  the 
substance  of  the  nail 
(fig.  312),  and  under  it  a series  of  transverse  sections  of  the  ridges 
of  the  corium  of  the  nail-bed  projecting  into  the  epidermis.  Under 
this  the  dense  fibrous  matrix. 

16.  L.S.  Nail. — Observe  the  same  general  arrangement,  but  note 
that  no  papilla-like  sections  of  ridges  are  to  be  seen. 


Pig.  312.— T.S.  Through  Half  the  Nail,  Injected,  a.  Nail 
substance ; h.  More  open  layer  of  cells ; c.  Stratum 
Malpighii;  d.  Transverse  sections  of  ridges;  e.  Nail- 
groove  ; /.  Horny  layer  of  e projecting  over  the  nail ; 
g.  Papillae  of  skin. 


ADDITIONAL  EXERCISES. 


17.  Elastic  Fibres  in  the  Skin. — (i.)  These  resist  gastric  digestion  ; hence,* 
add  some  pepsin  to  .2  per  cent,  of  hydrochloric  acid.  If  small  pieces  of  skin 
be  partially  digested  in  artificial  gastric  juice  at  40°C.,  part  of  the  connective 
tissue  is  dissolved  and  the  elastic  networks  remain. 

(ii.)  Herxheimer’s  Method. 


(1.)  Harden  in  Muller’s  fluid. 
(2.)  Stain  3-5  minutes  in 

Haematoxylin 
Absolute  alcohol 
Water 

Sat.  sol.  lithic  carb.  . 


I gram. 
20  cc. 

20  ,, 

I „ 


XXIX.] 


THE  NAILS. 


327 


(3.)  Extract  (5-15  secs.)  with  official  perchloride  of  iron  solution, 

(4.)  Wash  in  water. 

(5.)  Alcohol,  oil,  balsam. 

The  elastic  fibres  are  bluish-black  or  black,  and  the  surrounding  tissue  light 
blue.  What  Herxheimer  described  as  “spirals”  in  the  lower  layer  of  the 
epidermis  are  spiral  fibrils  proceeding  from  the  lowest  layer  of  the  cells  of  the 
epidermis,  as  shown  by  Kronmayer  (p.  327). 

(iii.)  Unna’s  Method. — Place  sections  of  the  skin  for  12-24  hours  in  the 
following  mixture : — 


Orcein 

Absolute  alcohol 
Water 

Hydrochloric  acid 


0.5  gram. 
40  cc. 

20  ,, 

20  drops. 


The  sections  are  afterwards  decolorised  in  very  dilute  HCl  containing  some 
alcohol.  A full  account  of  the  arrangement  of  the  elastic  fibres  is  given  by 
Lenthoefer.^ 

18.  Sweat-Glands  of  Axilla. — Make  Y.S.  of  the  skin  of  the  axilla  hardened 
in  alcohol.  Stain  in  hsematoxylin,  or  stain  in  bulk  and  cut  in  paraffin.  The 
sweat-glands,  and  particularly  the  coils,  are  very  large.  In  these  glands  it  is 
easy  to  see  the  smooth  muscular  fibres  outside  the  lining 

epithelium  of  the  secretory  part  of  the  coil  (fig.  313). 

19.  Development  of  Hairs. — Make  V.S.  of  the  skin  of 

a foetus  at  the  fourth  to  the  fifth  month,  after  being 
hardened  in  Muller’s  fluid  (12-15  and  then  in 

alcohol.  Stain  a small  piece  “ in  bulk  ” in  borax-car- 
mine, and  cut  in  paraffin.  The  sections  may  also  be 
stained  subsequently  with  methyl-green.  Mount  in 
balsam. 

20.  Double-Staining  of  Hair- Follicles. — (a.)  Make  V. 
and  T.  sections,  and  stain  some  with  eosin  and  hfemato- 
xylin,  and  others  first  with  picro-carmine  (12  hours)  and 
then  with  methyl-green  iodide.  The  latter  preparation 
is  specially  beautiful,  and  both  T.S.  and  L.S.  should  be 
stained  by  this  method.  The  scalp  is  best  hardened  in  potassic  bichromate. 
Harden  other  pieces  in  alcohol.  Methyl-green  stains  the  inner  root-sheath 
green. 

(h. ) Stain  with  aniline-blue  and  safranin.  Henle’s  sheath  is  rosy,  Huxley’s 
blue. 

(c. ) Or  stain  in  safranin  in  a weak  alcoholic  solution  of  picric  acid. 

21.  Tactile  Hairs.  — Harden  the  skin  containing  the  large  tactile  hairs  of 
a rabbit  or  cat  in  alcohol  or  Miiller’s  fluid.  Make  T.S.  and  L.S.  Outside  the 
sheaths  of  the  hair- follicle,  already  described,  there  is  a large  blood-space 
traversed  by  trabeculse,  and  thus  presenting  the  characters  of  cavernous 
tissue. 

22.  Nail  {double-stained). — Picro-carmine  and  methyl-green. 

23.  Blood-Pigment  in  Hair  (H). — Examine  in  normal  saline  one  of  the 
large  “ feelers”  from  the  lip  of  an  albino  rabbit  (aS'.  Mayer).  At  some  part  of 
the  hair  in  its  centre  a red  pigment — hfemoglobin — may  be  seen. 

24.  Fibrillation  of  Protoplasm  of  Epithelial  Cells  {Kronmayer)."^  By  means 
of  Weigert’s  fibrin-staining  method  (Lesson  III.  20)  Kronmayer  has  demon- 


FiG.  313.— T.S.  Secre- 
tory Part  of  Sweat- 
Gland  of  Axilla,  a. 
Nuclei  of  smooth 
muscle. 


^ Topographie  d.  ElastiscJien  Gewehe,  Leipzig,  1892. 

2 “Die  Protoplasmafaserung  d.  Epithelzelle,”  Archiv  /.  mik.  Anat.^  xxxix. 
p.  141,  1892. 


328 


PRACTICAL  HISTOLOGY. 


[xxix. 


strated  the  passage  of  the  fibrils  of  one  epithelial  cell  into  adjacent  epithelial 
cells,  thus  giving  rise  in  the  intercellular  spaces  to  the  appearances  known  as 
“intercellular  bridges  ” (p.  128).  The  fresh  skin  of  the  sole  of -the  foot  or 
palm  of  the  hand  is  hardened  in  absolute  alcohol.  Failing  this,  use  part  of  an 
epithelioma.  It  may  be  stained  with  alum-carmine  or  borax-carmine,  and  then 
embedded  and  cut  in  paraffin.  The  sections  must  be  very  thin  (at  h-ast  0.005 
mm.),  and  in  order  to  obtain  these  the  knife  must  be  placed  obliquely  and  the 
sections  cut  from  the  epithelial  surface  towards  the  cutis  vera. 

The  sections  are  jdaced  in  a watch-glass  and  xylol  added  to  dissolve  the 
paraffin.  Add  fresh  xylol,  pour  it  off’,  and  then  slowly  add  absolute  alcohol. 
Gradually  add  water  until  the  sections  float  flat  on  the  surface.  They  are  then 
carefully  transferred  to  a slide,  and  by  the  aid  of  a pad  of  filter-paper  gently 
fixed  on  the  same.  The  sections  so  fixed  are  stained  on  the  slide. 

The  best  staining  reagent  is  methyl- violet-6B,  prepared  as  follows  : — Mix 
equal  parts  of  aniline- water  and  a saturated  watery  solution  of  methyl  violet. 
Pour  a few  drops  of  this  fluid  on  the  section  fixed  on  the  slide,  and  in  five 
minutes  lave  the  section  in  water. 

Decolorise  (few  seconds)  in  iodine  solution  (p.  93).  Wash  in  water.  Remove 
the  water  by  pressing  blotting-paper  on  the  section,  and  then  flood  it  with 
aniline-xylol  (aniline  oil,  i : xylol,  2).  This  mixture  extracts  the  surplus 
dye  very  rapidly,  so  that  the  preparations  must  remain  but  a few  seconds 
therein. 

The  march  of  events,  supposing  one  wishes  to  stain  the  cell  nuclei  before 
staining  the  protoplasm  fibrils,  is  as  follows — the  cells  being  previously 
hardened  : — 


(i.)  Alum-carmine. 

(2.)  Wash  in  water  (HCl-alcohol, 
absolute  alcohol). 

(3. ) Methyl-violet-aniline-water. 

(4.)  Wash  in  water. 


(5.)  lodo-potassic-iodide  fluid. 
(6.)  Wash  in  water. 

(7.)  Aniline-xylol. 

(8.)  Xyloi-balsam. 


LESSON  XXX. 

SPINAL  CORD. 

The  spinal  cord,  like  the  brain,  is  invested  by  three  membranes, 
named,  from  without  inwards,  dura,  arachnoid,  and  pia  mater. 
The  pia  mater  closely  invests  the  cord,  and  sends  processes  into  its 
substance  as  well  as  into  its  fissures.  The  cord  itself  is  composed 
of  white  matter  externally  and  grey  matter  internally.  Running 
along  the  cord  anteriorly  and  posteriorly  are  the  anterior  and 
posterior  median  fissures;  the  former  is  the  wider,  the  latter 
rather  a groove  than  a fissure.  The  two  fissures  do  not  meet  in  the 
middle  line,  but  they  serve  to  divide  the  cord  incompletely  into  two 
lateral  halves,  which  are  united  across  the  middle  line  by  a com- 


XXX.] 


BPINAL  CORD. 


329 


missure,  composed  anteriorly  of  white  fibres  crossing  from  one  side 
of  the  cord  to  the  other — white  commissure ; and  posteriorly  of 
grey  matter — posterior  commissure.  In  the  middle  of  the  latter 
is  the  minute  central  canal,  lined  by  columnar  ciliated  epithelium. 
Ill  each  half  of  the  cord  is  a crescent-shaped  mass  of  grey  matter 
as  seen  in  transverse  section,  the  two  masses  cojinecte  1 across  the 
middle  line,  and  presenting  more  or  less  the  form  of  an  H,  with  the 
extremities  of  its  vertical  limbs  turned  outwards.  Its  extremities 
are  the  anterior  and  posterior  cornua.  The  anterior  cornua  are 
generally  wider  and  shorter  than  the  posterior,  which  are  narrow, 
and  come  nearer  the  surface  of  the  cord.  The  nerve-roots  arise 
from  the  cornua,  the  anterior  root  by  several  bundles  from  the 
anterior  cornu,  and  the  posterior  root  by  a single  bundle  from  the 
posterior  one.  In  this  way,  and  by  the  existence  of  the  fissures, 
each  half  of  the  white  matter  of  the  cord  may  be  conveniently 
described  as  divided  into  an  anterior,  lateral,  and  posterior 
column,  or  more  correctly  into  an  antero-lateral  and  a posterior. 
The  anterior  cornu  contains  numerous  large  multipolar  nerve-cells 
arranged  in  groups.  Each  cell  is  continuous,  through  its  axis- 
cylinder  process,  with  a nerve-fibre.  The  arrangement  and  number 
of  cells,  however,  vary  in  different  parts  of  the  cord.  There  are  no 
large  nerve-cells — only  small  fusiform  ones  and  some  small  isolated 
or  “ solitary  ” cells — in  the  posterior  cornu,  which  is  capped  by  a 
peculiar  greyish  matter — the  substantia  gelatinosa  of  Kolando. 
The  white  matter  is  composed  of  medullated  nerve-fibres — small 
and  large — arranged  for  the  most  part  longitudinally,  so  that  in  a 
transverse  section,  when  stained  with  carmine,  they  appear  like 
clear  rings  with  a central,  red-stained  spot — the  axis-cylinder.  In 
PalVmethod  the  myelin  is  stained,  and  so  they  appear  as  blackish 
circles  with  a clear  centre.  The  nerve-fibres,  and  grey  matter  as 
well,  are  supported  by  a peculiar  sustentacular  tissue — neuroglia — 
composed  of  glia-cells  (p.  343),  and  both  are  supplied  by  blood- 
vessels, the  grey  matter,  however,  being  far  more  vascular  than  the 
white.  The  grey  matter,  besides  blood-vessels,  lymphatics,  glia-cells, 
nerve-cells,  and  their  numerous  processes,  also  contains  nerve-fibres. 

The  nerve-cells  vary  in  size,  shape,  and  other  characters  in  the 
different  parts  of  the  cord.  Golgi  speaks  of  two  types  of  nerve-cells 
in  the  spinal  cord. 

Type  I,  or  Motor  Type,  corresponding  to  the  multipolar  cells  of  the 
anterior  cornu.  In  these  one  process  retains  its  individuality,  and 
passes  directly  to  become  an  axis-cylinder  of  a medullated  motor 
nerve-fibre  in  the  anterior  root.  This  axis-cylinder  process  gives  off 
a few  secondary  lateral  processes  or  collaterals,  which  divide  and 
enter  into  the  formation  of  the  nerve-complex  in  the  grey  matter. 
The  protoplasmic  processes  subdivide,  and  also  form  part  of  the 


PRAC'JICAL  HISTOLOGY. 


330 


[xxx. 


same  grey  complex,  but  they  do  not  unite  with  fibres  from  adjoining 
cells  or  with  nerve-fibres. 

Type  2,  or  Sensory  Type,  Also  multipolar,  smaller  cells,  with  no 
axis-cylinder  process.  All  the  processes  subdivide  into  finer  pro- 
cesses, lose  their  individuality,  and  pass  in  toto  into  the  nervous 
complex,  or  diffuse  nervous  network  in  all  strata  of  the  grey  matter 
of  the  cord.  These  cells  are  therefore  connected  only  indirectly 
with  nerve-fibres,  i.e.^  through  the  intervention  of  the  grey  nervous 
network  ; and  it  is  only  in  this  indirect  way  that  they  come  into 
relation  with — contact,  not  actual  union  with — the  branches  of 
axis-cylinders  of  the  nerve-fibres  of  the  posterior  root. 

Arrangement  of  Nerve-Cells. — In  the  grey  matter  of  the  anterior 
cornu,  the  large  mvdtipolar  nerve-cells — cells  of  the  anterior  cornu — 
are  arranged  in  groups  varying  in  different  parts  of  the  cord.  Many 
of  their  axis-cylinder  processes  pass  out  as  axis-cylinders  into  the 
nerves  of  the  anterior  root  on  the  same  side  of  the  cord.  These 
cells  are  in  relation  with  the  fibrillar  nerve-endings  of  the  fibres  of 
the  pyramidal  tracts,  and  also  with  the  collateral  fibres  of  the  pos- 
terior root-fibres.  In  the  upper  dorsal  and  lower  cervical  regions 
is  a group  of  nerve-cells — intermedio-lateral  tract — lying  well  for- 
ward in  a projecting  part  of  the  grey  matter  known  as  the  lateral 
cornu  (fig.  317,  Til).  In  the  middle  of  the  crescent  is  the  middle 
cell  group.  At  the  base  of  the  posterior  cornu,  on  its  inner  aspect, 
is  a group  of  large  cells — best  marked  in  the  thoracic  region — 
Cdarkds  column  (figs.  318,  319,  (7(7/).  The  axis-cylinder  pro- 
cesses of  the  cells  of  Clarke’s  column  pass  into  and  become  the 
nerve-fibres  of  the  direct  cerebellar  tract  (p.  331).  The  cells  of  the 
posterior  cornu  are  small,  and  for  the  most  part  isolated. 

Dilfuse  Nervous  Grey  Network. — The  network  or  complex  of 
nervous  fibrils  in  the  grey  matter  of  the  cord  is  formed  by — (i.) 
the  fibres  and  their  branches  of  the  2nd  type  of  nerve-cells  (p.  330)  ; 
(2.)  the  fibrils  and  prolongations  of  the  protoplasmic  fibres  of  the 
ist  type;  (3.)  the  lateral  branches  of  the  axis-cylinder  processes  of 
the  ist  type  ; (4.)  the  fibrils  produced  by  the  terminal  arborisations  of 
the  axis-cylinders  entering  the  cord  by  the  posterior  roots. 

Thus  it  is  evident  that  some  nerve-fibres  spring  from  the  cord 
directly  from  nerve-cells,  and  others  indirectly  from  the  nervous 
complex  in  the  grey  matter.  Thus  they  are  different,  both  morpho- 
logically and  physiologically. 

By  several  lines  of  research — including  the  facts  of  development, 
experimental  and  pathological  evidence — the  anatomical  columns  of 
the  cord  can  be  shown  to  be  further  subdivided.  By  these  methods 
it  is  found  that  the  posterior  column  consists  of  a narrower  internal 
part,  the  postero  internal  or  postero-mesial  column,  or  funiculus 
gracilis  or  column  of  Goll,  and  an  outer,  postero-lateral  tract  or 


XXX.] 


SPINAL  CORD. 


331 


funiculus  cuneatus,  or  column  of  Burdach.  In  the  upper  part  of  the 
cord  these  two  tracts  are  mapped  off  from  each  other  by  a septum 
of  connective  tissue  (fig.  317). 

postero-internal  tract  is  composed  chiefly  of  rather  small  fibres, 
derived  from  the  fibres  of  the  posterior  roots  and  fibre  of  the  pos- 
tero-lateral  column.  They  end  above  grey  matter  in  the  nucleus 
gracilis  of  the  bulb. 

The  postero-lateral  tract  is  chiefly  composed  of  nerve-fibres  of  the 
posterior  roots  which  run  in  it  a certain  distance  before  they  pass 
into  the  grey  matter  and  Golhs  column.  They  end  in  grey  matter 
in  the  cord  and  in  the  nucleus  cuneatus  of  the  bulb. 

In  the  postero-lateral  column  is  a small  zone  which  undergoes 
descending  degeneration,  but  only  for  a short  distance,  after  section 
of  the  cord — the  so-called  comma  tract. 

Lissauer^s  Tracts  or  ‘‘  marginal  bundle f lies  near  the  entrance  of 
the  posterior  roots,  eitlier  in  the  lateral  column  or  postero-external 
column.  It  is  derived  directly  from  the  fibres  of  the  posterior  roots. 
It  undergoes  ascending  degeneration. 

The  antero-lateral  column  contains  a large  tract — the  crossed 
pyramidal  tract — which  lies  external  to  the  posterior  half  of  the 
grey  matter,  and  in  the  greater  part  of  its  course  is  separated  from 
the  surface  of  the  cord  by  the  direct  cerebellar  tract.  It  consists 
of  fibres  descending  from  the  central  areas  (motor J of  the  cerebral 
cortex,  which  have  crossed  at  the  decussation  of  the  pyramids  in 
the  bulb.  It  consists  of  moderately  large  and  some  small  fibres. 
Its  fibres  end  by  breaking  up  into  fibrils,  which  form  arborisations 
around  the  nerve-cells  in  the  anterior  cornu.  It  gradually 
diminishes  in  size  as  it  passes  down  the  cord,  and  can  be  traced  as 
far  as  the  origin  of  the  3rd  or  4th  sacral  nerve,  where  it  reaches  the 
surface. 

The  direct  pyramidal  tract  or  column  of  TiXrcU  bounds  the 
anterior  median  fissure,  and  consists  of  fibres  coming  from  the 
motor  areas  of  the  cerebral  cortex,  which  do  not  cross  at  the  bulb. 
This  tract  gets  smaller,  and  gradually  disappears  about  the  mid- 
dorsal region.  Its  fibres  perhaps  cross  in  the  cord.  These  two  are 
descending  tracts. 

Beginning  at  the  lower  dorsal  region,  and  increasing  in  size  as  it 
passes  upwards,  is  a 4ract  lying  external  to  the  crossed  pyramidal 
tract — the  direct  cerebellar  or  dorso-lateral  tract — which  con- 
sists of  large  fibres  derived  from  the  cells  of  Clarke’s  column, 
which  pass  up  to  the  cerebellum,  and  enter  it  on  the  same  side  by 
its  inferior  peduncle  or  restiform  body.  Near  the  surface  of  the 
cord,  and  lying  more  anteriorly,  is  a tract  which  extends  ventrally 
into  the  anterior  column — the  antero-lateral  ascending  tract  of 
Gowers.  Sometimes  the  term  ventro-lateral  is  applied  instead  of 


PRACTICAL  HISTOLOGY. 


332 


[xxx. 


antero-lateral.  It  enters  the  cerebellum  by  the  superior  cerebellar 
peduncle. 

In  the  dog,  as  a result  of  excision  of  one-half  of  the  cerebellum, 
a circumferential  tract,  occupying  three-fourths  of  the  surface  of  the 
antero-lateral  tract — the  antero-lateral  descending  cerebellar  trad — 
has  been  mapped  out.  The  remainder  of  the  antero-lateral  column 
is  spoken  of  as  the  antero-lateral  ground  bundle. 

As  regards  the  results  of  degeneration  following  section  or  lesion 
of  the  cord,  those  parts  that  undergo  degeneration  above  the  lesion 
are  called  “ascending  tracts’^;  and  “descending  tracts,^’  are  those 
nerve-fibres  that  degenerate  below  the  lesion  or  seat  of  section. 
The  parts  which  may  undergo  these  respective  degenerations  are  : — 

f Direct  pyramidal  tract. 

Descending  J Crossed  pyramidal  tract. 

degeneration.  1 Antero-lateral  descending  cerebellar  tract  (limited). 

I Comma  tract, 
f Goll’s  column. 

Ascending  J Direct  cerebellar  tract. 
degeneration,  j Tract  of  Gowers. 

t Tract  of  Lissauer. 

It  will  thus  be  seen  that  in  the  antero-lateral  columns  there  are 
some  nerve-fibres  which  do  not  undergo  degeneration. 

Course  of  Fibres  of  Nerve-Eoots. — The  fibres  of  the  anterior 
roots  arise  in  several  bundles  from  the  axis-cylinder  processes  of  the 
multipolar  nerve-cells  in  the  anterior  cornu. 

The  fibres  of  the  posterior  roots  enter  the  cord  by  a single  bundle, 
but  each  one  is  an  axial  cylinder  process  of  a nerve-cell  in  the 
corresponding  spinal  ganglion.  They  pass  into  the  postero-lateral 
column,  some  pass  into  the  posterior  cornu,  and  a few  small  fibres 
form  the  marginal  bundle  (p.  331).  Many  fibres  pass  up  in  GolFs 
column,  and  the  postero-external  tract  to  end  in  terminal  arborisa- 
tions of  fibrils  around  nerve-cells  in  the  bulb,  the  fibres  of  GolFs 
column  in  arborisations  around  the  cells  of  the  nucleus  gracilis,  and 
those  of  the  postero-external  tract  in  the  nucleus  cuneatus.  After 
entering  the  cord,  the  fibres  in  the  posterior  columns  bifurcate,  one 
branch  passes  upwards  and  one  downwards  (fig.  322).  Collateral 
fibres  are  given  off  from  the  original  fibre,  and  also  from  its 
branches,  which  enter  the  grey  matter  and  end  by  terminal  arbori- 
sations of  fine  fibrils  which  come  into  relation*-but  not  direct  union 
— with  the  nerve-cells  of  the  grey  matter,  notably  with  the  cells 
of  Clarke’s  column. 


THE  SPINAL  COED. 

It  is  convenient  to  begin  with  the  cord  of  a small  animal,  e.g.^  a 
cat  or  dog,  but  the  student  must  also  be  provided  with  sections  of 


SPINAL  CORD. 


333 


XXX.] 

the  human  cord.  The  same  methods  are  applicable  to  both. 
Remove  the  whole  length  of  the  spinal  cord  from  a cat,  taking  care 
not  to  squeeze  or  crush  it  in  the  process.  Make  transverse  cuts 
into  it  about  f of  an  inch  apart,  and  suspend  it  in  a tall  vessel  in  a 
large  quantity  of  Muller’s  fluid,  or  2 per  cent,  ammonium  or 
potassium  bichromate.  Keep  it  in  a cool  place  in  the  dark. 
Bichromate  of  ammonium  hardens  the  cord  very  slowly  indeed. 
In  fact,  to  get  a properly  hardened  cord  months  are  required.  To 
test  if  the  cord  is  properly  hardened,  cut  a section  of  the  cord  taken 
from  the  ammonium  bichromate  fluid,  place  it  in  water,  and  if  it 
curls  up,  it  is  not  properly  hardened.  It  ought  to  remain  flat. 
The  process  may  be  expedited  by  hardening  first  for  4 or  5 w^eeks 
in  the  bichromate,  and  completing  the  hardening  for  2-3  weeks  in 
i per  cent,  chromic  acid.  Change  the  hardening  fluid  on  the 
second  day,  and  repeatedly  thereafter.  After  4-5  weeks,  when  it 
becomes^  tough,  wash  it,  and  harden  in  the  various  strengths  of 
alcohol,  beginning  with  50  per  cent.  If,  however,  the  spinal  cord 
is  to  be  used  for  Weigert’s  haematoxylin  stain,  it  must  not  be 
washed  in  water,  but  placed  in  alcohol  direct  from  the  Muller’s 
fluid  or  potassic  bichromate. 

T.S.  are  made  from  the  cervical,  dorsal,  and  lumbar  regions. 
They  may  be  made  by  means  of  the  freezing  microtome,  the  cord 
having  been  previously  saturated  in  the  sugar  and  gum  mixture ; 
or  small  pieces  of  the  cord  may  be  stained  in  borax-carmine 
(i  week),  and  then  cut  in  paraffin.  Or  they  may  be  embedded  and 
cut  in  celloidin,  and  afterwards  stained  (p.  60).  The  paraffin 
sections  are  fixed  to  a slide  by  a “fixative,”  the  paraffin  removed 
by  turpentine  or  xylol,  the  sections  clarified  by  clove-oil,  and 
mounted  in  balsam.  Sections,  if  made  by  freezing,  may  be  stained 
with  carmine,  hsematoxylin,  aniline-blue-black,  safranin,  methylene- 
blue,  or  by  other  methods. 

1.  T.S.  Spinal  Cord  of  Cat  (L)  and  (H). — Speaking  broadly, 
the  same  general  arrangement  obtains  as  in  fig.  314.  Suppose  it  to 
be  the  dorsal  region,  and  the  cord  to  be  stained  in  carmine  and 
mounted  in  balsam,  observe  : — 

(a.)  (L)  The  nearly  circular  outline  of  the  section,  covered 
externally  by  the  pia  mater,  composed  of  two  layers.  From  its 
under  surface  septa  run  into  the  white  matter  of  the  cord,  and  some 
of  them  carry  blood-vessels,  and  processes  pass  into  the  fissures  of 
the  cord. 

{h.)  The  anterior  and  posterior  median  fissures.  The 
anterior  fissure  is  wider  and  better  marked.  In  it  run  both 
layers  pf  the  pia  mater.  The  posterior  fissure  appears  as  a 
septum  due  to  the  prolongation  of  the  inner  layer  of  the  pia  mater 
into  it,  the  outer  layer  of  the  pia  runs  over  the  fissure.  The 


PRACTICAL  HISTOLOGY. 


334 


[xxx. 


cord  is  thus  almost  completely  divided  into  two  symmetrical  halves 

(fig-  314)- 


(c.)  If  the  section  is  made  at  the  level  of  the  origin  of  the 
nerve-roots,  these  may  he  seen.  The  mode  of  origin  of  some  of  the 
fibres  of  these  roots  can  always  be  seen.  The  anterior  root  passes 


Fig.  314. — T.S.  Lower  Dorsal  Cord,  Human.  A.  L,  P,  Anterior,  lateral,  and  posterior 
columns;  A.M.F , P.M.F.  Anterior  and  posterior  median  fissures;  a,  b,  c.  Nerve- 
cells  of  the  anterior  horn;  d Posterior  cornu  and  substantia  gelatinosa;  e.  Central 
canal;/.  Veins,  g.  Origin  of  anterior  nerve-root;  h.  Posterior  nerve-root ; i.  White, 
and/  Grey  commissures;  1.  Reticular  formation. 

out  of  the  cord  in  several  bundles,  while  the  posterior  root  enters 
the  cord  in  one  compact  bundle. 

{(1.)  The  white  matter  externally,  and  the  grey  matter  internally, 
the  latter  deeply  stained.  The  origin  of  the  nerve-roots  dividing 
the  white  matter  into  the  anterior,  lateral,  and  posterior  columns, 
but  owing  to  the  anterior  root  leaving  the  cord  in  several  bundles, 
there  is  not  an  exact  anatomical  limitation  of  the  anterior  from  the 
lateral  column. 

{e.)  The  grey  matter,  deeply  stained — a crescentic  mass  in  each 
half  of  the  cord,  with  a broader  anterior  cornu,  with  numerous 


XXX.] 


SPINAL  CORD. 


335 


large  multipolar  nerve-cells,  arranged  in  groups — which  does  not 
reach  the  surface  of  the  cord,  and  a 'posterior  cornu — with  a few 
small  nerve-cells — which  comes  to  the  surface,  and  is  prolonged  into 
the  posterior  root.  At  the  hinder  part  of  the  posterior  cornu,  an  oval, 
deeply  stained  part,  the  substantia  gelatinosa.  A neck  connecting 
the  two  cornua.  There  is  also  a 
group  of  nerve-cells  placed  laterally 
in  the  grey  matter,  the  vesicular 
column  of  Clarke  (fig.  318).  In 
the  anterior  horn,  note  especially 
the  entrance  of  the  fibres  of  the 
anterior  nerve-root  and  the  large 
multipolar  nerve-cells  (fig.  315). 

(/.)  Connecting  the  two  halves 
of  the  cord,  the  anterior  and 
posterior  commissures,  running 
between  the  grey  matter  of 
opposite  sides,  and  in  the  middle 
between  them  the  central  canal, 
which  is  surrounded  externally 
by  a deeper  stained  layer  of 
neuroglia. 

{g.)  In  front  of  the  anterior 
grey  commissure  is  the  anterior 
white  commissure,  with  large  medullated  fibres  crossing  each  other 
at  an  angle. 

{h.)  (H)  The  pia  mater  surrounding  the  white  matter.  The  cut 
ends  of  the  medullated  nerve-fibres  of  various  sizes.  In  the  centre 
of  each  circular  area  the  stained 
axis-cylinder,  surrounded  by  a 
concentric  clear  area — the  white 
substance  of  Schwann  (fig.  316). 

In  a preparation  hardened  in 
chromic  acid  not  infrequently  a 
number  of  concentric  lines  are 
seen  in  the  myelin.  Between 
the  fibres  here  and  there  the 
neuroglia,  composed  of  glia  cells. 

On  the  surface  of  the  cord  there 
is  a thin  layer,  in  which  the  fibrils 
of  the  neuroglia  can  readily  be  seen.  iN'ote  also  the  prolonga- 
tions, as  fine  septa,  of  the  deeper  part  of  the  pia  into  the 
substance  of  the  cord.  The  larger  septa  carry  blood-vessels. 
The  nerve-fibres  of  the  antero-lateral  columns  are  generally 
larger,  ix.,  broader,  than  those  of  the  posterior  column,  while  those 


Fig.  315.— Entrance  of  Anterior  Root  into 
the  Anterior  Cornu  of  Lumbar  Region. 
I.  Part  of  anterior  white  column  ; 2. 
Anterior  grey  matter  with  four  multi- 
polar cells  ; a,  a'.  Two  rootlets,  x 30. 


White  Matter  of  Cord. 
a.  Periplieral  layer ; h.  Septum ; c. 
Branched  glia-cell ; the  remainder 
nerve-fibres,  small  and  large,  x 150. 


PRACTICAL  HISTOLOGY. 


336 


[xxx. 


of  the  column  of  Goll  are  remarkable  for  their  small  size.  Measure 
the  sizes. 

(^.)  The  anterior  cornu,  with  large  multipolar  nerve-cells  arranged 


Fig.  317.— T.S.  of  Human  Spinal  Cord,  Level  of  FlQ.  318.— At  the  Level  of  the 

Sixth  Cervical  Nerve.  Prm.  Middle  cervical  Third  Dorsal  Nerve, 

process  of  the  anterior  cornu ; Til.  Lateral 
horn. 


in  groups,  chiefly  in  the  anterior  and  lateral  parts  of  the  cornu. 
Each  cell  has  numerous  processes,  a spherical  well-defined  nucleus 
with  a membrane  and  a nucleolus  (fig.  202).  Owing  to  the  method 


Fig.  319. — At  the  Twelfth 
Dorsal  Nerve.  CCl.  Clarke’s 
column. 


Fig.  320. — Level  of  Fifth  Lumbar 
Nerve.  m.  Median  group  of 
nerve-cells  of  anterior-cornu ; 
IVy  Id,  and  c.  Latero- ventral, 
latero-dorsal,  and  central  groups 
of  cells,  X 5. 


of  hardening,  all  the  cells  are  somewhat  retracted,  each  cell  ap- 
parently lying  in  a cavity. 

(J.)  Trace  medullated  nerve-fibres  across  the  white  commissure 


SPINAL  CORD. 


XXX.] 


337 


from  the  grey  matter  of  one  side  to  the  white  matter  of  the  opposite 
side. 

(k.)  The  posterior  cornu.  [N'ote  the  absence  of  large  cells. 
Only  a few  small  fusiform  nerve-cells  are  seen.  Some  of  the  fibres 
of  the  posterior  root  pass  into  the  posterior  cornu ; some  pass 
directly  through  the  substantia  gelatinosa,  and  others  sweep  with  a 
curve  through  the  posterior  column  before  they  enter  the  grey 
matter.  The  grey  matter  generally  is  traversed  by  fine  fibrils,  and 
has  a somewhat  finely  granular  appearance-o 

(/.)  The  central  canal,  lined  by  a single  layer  of  columnar 
ciliated  epithelial  cells.  The  cilia  may  be  wanting.  Near  it  sec- 
tions of  blood-vessels.  In  the  grey  matter  observe  the  plexus  of 
fine  fibrils  and  numerous  axis-cylinders.  (Multipolar  nerve-cells, 
Lesson  XYIII.  9.) 

2.  T.S.  Human  Spinal  Cord  in  several  Regions. — Stain  sec- 
tions with  carmine  or  lithium-carmine,  hsematoxylin,  or  aniline 
blue-black,  and  mount  in  balsam.  Mount  sections  from  the  cervical 
enlargement  (fifth  nerve)  and  lumbar  enlargement,  and  compare 
them  with  the  dorsal  section.  With  the  naked  eye  (and  L) 
observe : — ■ 

(a.)  The  cervical  and  lumbar  sections  (figs.  317,  320)  are  not 
only  larger,  but  the  amount  of  grey  matter  is  greater  than  in  the 
dorsal  region.  Note  the  large  expanded  anterior  cornu  with 
numerous  nerve-cells.  In  the  lumbar  region  the  grey  matter  is 
large  in  amount,  the  white  matter  smallest.  The  white  matter  is 
more  abundant  in  the  dorsal  region  and  most  abundant  in  the 
cervical  region.  This  can,  to  a certain  extent,  be  determined  by 
the  amount  of  white  matter  lying  between  the  grey  matter  and 
the  pia  mater.  There  is  a gradual  transition  from  the  one  region 
of  the  cord  to  the  other. 

(6.)  In  the  cervical  region  note  a thin  septum,  which  dips  into 
the  white  matter  and  divides  its  posterior  column  into  a smaller 
internal  part — the  postero  internal  column  or  column  or  fasciculus 
of  GoU,  and  a larger  external  or  lateral  part — the  postero-external 
column,  or  fasciculus  cuneatus.  The  grey  matter,  containing  some 
nerve-cells,  also  projects  into  the  white  matter  about  midway 
between  the  anterior  and  posterior  cornua,  forming  the  intermedio- 
lateral  tract, 

{c.)  In  the  dorsal  region,  observe  a group  of  nerve-cells  at  the 
inner  part  of  the  neck  of  the  grey  matter.  It  lies  behind  the  plane 
of  the  central  canal — column  of  Clarke,  ov  posterior  vesicular  column 
(fig.  319,  ecz). 

In  sections  of  the  cord  at  different  levels  it  is  important  to  note 
the  sectional  area  of  the  difierent  columns.  Thus,  Goll’s  column  is 
largest  in  the  upper  cervical  region,  and  diminishes  from  above 
30  Y 


338  PRACTICAL  HISTOLOGY.  [XXX. 

downwards.  The  increase  or  decrease  in  the  sectional  area  of  some 
other  columns  can  only  he  made  out  by  a study  of  degenerated  or 
developing  cords. 

3.  L.S.  of  Cord  (Anteroposterior  direction). — Stain  as  for 
T.S.  (L  and  H.) 

(a.)  Observe  the  longitudinally-arranged  nerve-fibres  in  the  white 
matter  of  the  anterior  and  posterior  columns.  In  the  anterior 
cornu  the  rows  of  multipolar  nerve-cells. 

(b.)  The  anterior  roots  passing  obliquely  through  the  anterior 
column. 

(c.)  The  posterior  cornu,  with  its  gelatinous  substance  and  nerve- 
fibres  passing  into  and  through  it. 

4.  Weigert’s  Haematoxylin  Methods — The  myelin  of  medul- 
lated  fibres  is  stained  of  a deep  blue-black  tint,  while  degenerated 
parts  are  not  so  stained;  they  are  lighter  just  in  proportion  to 
the  disappearance  of  medullated  fibres.  The  spinal  cord,  or  the 
brain,  is  hardened  for  four  to  six  weeks  in  Muller’s  fluid,  and  the 
hardened  pieces  are  placed  direct  into  70  per  cent,  alcohol,  and 
must  not  be  washed  with  water.  Keep  in  the  dark.  Kext  day 
add  90  per  cent,  alcohol,  and  harden  completely  in  95  per  cent,  or 
absolute  alcohol.  Instead  of  Muller’s  fluid,  Eriicki’s  fluid  may  be 
used.  This  is  best  done  by  keeping  the  fluid  at  the  temperature 
of  the  body  in  a warm  chamber,  when  the  hardening  process  is 
completed  in  a few  days;  but  the  results  are  not  so  satisfactory  as 
in  the  case  of  a cord  hardened  in  the  ordinary  way.  Small  pieces 
of  the  hardened  tissue  are  placed  in  a half-saturated  solution  of 
neutral  acetate  of  copper  (^.e.,  a saturated  solution  is  mixed  with 
its  own  volume  of  distilled  water),  where  they  remain  for  three  to 
five  days.  They  are  then  transferred  direct  to  90  per  cent,  alcohol, 
and  can  then  be  cut  in  alcohol,  or  the  pieces  can  be  embedded  in 
celloidin  and  then  cut  (p.  47). 

(A.)  The  sections  are  placed  for  two  or  three  hours  in  a few  cc.  of 
Weigert’s  haematoxylin,  in  which  they  become  black. 

Weigert’s  Hsematoxylin. 


j^Hsematoxyliii .....  i irrani. 

' (Alcohol  absolute  . . . . 10  cc. 

2 fCold  saturated  solution  of  lithium  carbonate  i ,, 

^ {Distilled  water  . . . 90  »» 


Dissolve  the  hsematoxylin  in  the  absolute  alcohol,  add  the  water 
and  boil.  After  it  is  cool,  add  the  lithium  carbonate.  The  time 
which  the  sections  remain  in  this  fluid  depends  on  what  it  is 

^ Fortschrit,  d.  Med.,  1884  and  1885 ; Zeits.  f.  wissensch.  Mik.,  1884  and 
1885. 


XXX.] 


SPINAL  CORD. 


339 


desired  to  show.  Two  hours  or  so  are  enough  for  the  cord,  but 
if  the  fine  plexuses  of  medullated  fibres — described  by  Exner — in 
the  cerebral  cortex  are  to  be  well  seen,  let  them  stain  for  twenty- 
four  hours.  After  they  are  sufficiently  stained,  throw  the  watch- 
glass  and  the  stai;ied  sections  into  a large  basin  of  distilled  water. 
Kemove  the  sections  from  the  water  at  once,  and  place  them,  for 
about  half  an  hour  or  more,  in  the  following  mixture  : — • 

Potassic  ferricyanide  . . . 2.5  grams. 

Borax  . , . , 2 ,, 

Water  .....  100  cc. 

This  fluid  decolorises  and  differentiates  the  black  sections.  The 
grey  matter  becomes  of  a brown  or  light-yellow  tint,  and  should 
remain  so  while  the  white  matter  becomes  violet.  The  sections 
must  remain  in  the  decolorising  fluid  until  the  deep  blue  or  violet- 
coloured  medullated  nerve-fibres  are  seen.  This  can  readily  be 
determined  after  a little  practice.  The  sections  are  then  washed 
^ in  water — in  which  they  can  be  kept  for  a considerable  time — 
transferred  to  90  per  cent.,  and  finally  to  absolute  alcohol,  clari- 
fied by  xylol  or  origanum  oil,  and  mounted  in  xylol-balsam.  This 
method  stains  the  medulla  or  myelin  of  the  nerves — especially  of 
the  central  nervous  system — of  a deep  blue  or  violet  tint,  while  the 
nerve-cells,  neuroglia,  and  axis-cylinders  are  not  stained.  The 
method,  however,  may  be  combined  with  staining  methods.  After 
the  organ  is  hardened  in  the  chromium  salt  and  alcohol,  small  pieces 
are  embedded  in  celloidin  and  cut  in  a microtome.  The  sections 
arc  placed  in  80  per  cent,  spirit — not  water.  The  celloidin  enables 
the  sections  to  be  readily  handled  and  transferred  to  a slide.  They 
are  then  stained  in  the  special  haematoxylin  fluid  already  described. 
If  a series  of  sections  is  to  be  mounted  on  the  same  slide,  see  the 
method  described  at  p.  60.  The  sections  are  clarified  in  origanum 
oil  or  a mixture  of  xylol  and  carbolic  acid  (p.  83).  Mounted  in 
balsam.  This  method  is  also  applicable  to  the  spinal  ganglia. 

(B.)  Freezing  Method. — The  piece  of  cord  or  brain,  after  harden- 
ing in  Muller’s  fluid,  is  placed  in  spirit  and  transferred  to  a mixture 
of  equal  parts  of  absolute  alcohol  and  ether,  and  then  embedded  in 
celloidin.  The  embedded  tissue  is  placed  for  forty-eight  hours  in 
Erlicki’s  fluid,  to  get  rid  of  the  spirit,  and  they  are  then  placed  in 
the  following  mixture,  and  kept  in  stoppered  bottles  in  a warm 
chamber  at  38°  C.  for  two  or  three  days  : — 

Cupric  sulphate  , ....  0.5  gram. 

Potassic  bichromate  ....  2.5  grams. 

Mucilage  of  syrup  and  gum  . . . 100  cc. 

Sections  are  cut  in  a freezing  microtome  and  received  into  Erlicki’s 
fluid,  washed  in  methylated  spirit,  stained  in  the  haematoxylin  fluid. 


PRACTICAL  HISTOLOGY. 


340 


[xxx. 


decolorised  by  the  ferricyanide  mixture,  clarified,  and  mounted  in 
balsam  {Hamilton). 

Stages  of  Weigert’s  Method. 

(1.)  Harden  nervous  system  in  Muller’s  fluid.  v 

(2.)  Harden  in  alcohol  without  previous  washing  in  water. 

(3.)  Embed  in  celloidin. 

(4.)  Place  celloidin  block  in  a half- saturated  solution  of  copper  acetate 
(24-48  hours). 

(5.)  Alcohol,  70  per  cent.  (24  hours). 

(6.)  Cut  sections  and  stain  them  in  Weigert’s  hiematoxylin  (24  hours). 

(7.)  Wash  in  water. 

(8.)  Partially  decolorise  in  ferricyanide  fluid  until  grey  matter  becomes 
yellow. 

(9.)  Wash  in  water,  dehydrate  in  absolute  alcohol,  clarify  in  xylol,  and 
mount  in  xylol-balsam. 

Pal’s  method  is  referred  to  on  p.  343,  but  the  following  combina- 
tion of  the  Pal  and  Weigert  method  gives  good  results  : — 

5.  Modified  Weigert-Pal  Method. 

(i.)  Harden  in  Muller’s  fluid  and  then  in  alcohol,  without  wash- 
ing pieces  in  water. 

(ii.)  Embed  and  cut  sections  in  celloidin. 

(iii.)  Wash  in  water  and  transfer  to  Marchi’s  fluid  (5-10  hours). 

(iv.)  Wash  and  transfer  for  10-16  hours  to 

Knltschitzky’s  Haematoxylin. 

Hematoxylin  .....  i gram. 

Absolute  alcohol  ....  2-5  cc. 

Dissolve,  and  to  the  fluid  add 

Acetic  acid  (2  per  cent.)  . . . 100  cc. 

The  sections  become  black. 

(v.)  Bleaching  process — (a.)  Wash  in  water  and  bleach  for  5 
mins,  in  .2  per  cent,  permanganate  of  potash.  Wash 
again,  and  transfer  to 

(h,)  Pal’s  solution  (p.  344),  in  which  they  are  rapidly  (5-10 
mins.)  bleached,  the  medullated  fibres  alone  remaining 
black. 

(vi.)  Wash  in  water,  dehydrate  in  alcohol,  balsam,  oil  (xylol), 
.balsam  {Schafer), 

6.  Weigert-Stained  Cord. — (a.)  (Land  H)  Observe  the  medulla 
of  the  medullated  nerves,  stained  purplish.  There  are  so  many  fine 
medullated  nerve-fibres  revealed  in  the  grey  matter  that  it  is  impos- 
sible to  reproduce  their  complexity  in  a woodcut  The  nerve-cells 
are  not  specially  in  evidence,  although  they  can  be  stained  with 
picro-carmine. 


XXX.] 


SPINAL  CORD. 


341 


Formerly  Weigert  used  acid-fuchsin  for  staining  the  medullated 
nerve-fibres  of  the  central  nervous  system,  but  this  method  is  now 
given  up  in  favour  of  the  hsematoxylin  copper  method. 

7.  Neuroglia  (H). — Stain  in  safranin  (24  hours)  thin  sections  of 
the  white  matter  of  the  cord  hardened  in  Muller’s  fluid,  partially 
decolorise  in  absolute  alcohol,  and  mount  in  balsam. 

(a.)  Observe  the  branching  neuroglia-cells  between  the  white 
nerve-fibres  (fig.  316,  c).  Tlie  connective-tissue  elements  have  a 
tint  more  towards  the  violet,  and  are  thus  difiPerentiated  from  the 
nervous  elements.  The  network  of  neuroglia-fibres  is  readily  seen 
near  the  surface  of  the  cord. 

8.  Blood-Vessels  of  the  Spinal  Cord. — Mount  a transverse  sec- 
tion of  the  cord,  with  its  blood-vessels  injected.  Cut  the  cord  in 
paraffin.  The  sections  must  be  rather  thick  (L  and  H).  Inject  the 
animal,  e,g.^  cat  or  rabbit,  from  the  aorta  with  a blue  or  red  mass. 
Harden  in  alcohol. 

(a.)  Observe  the  greater  vascularity  of  the  grey  matter  as  com- 
pared with  the  white.  A blood-vessel  may  be  seen  running  into  the 
anterior  median  fissure,  and  at  the  bottom  of  it  dividing,  and  giving 
a branch  to  each  mass  of  grey  matter. 

{b,)  The  dense  plexus  of  capillaries  in  the  grey  matter.  Branches 
of  blood-vessels  passing  into  the  cord  along  the  roots  of  the  nerves 
and  along  the  larger  septa,  which  pass  from  the  pia  mater  into  the 
cord. 

9.  Nerve-Fibres  of  the  Spinal  Cord  (H). — Crush  a piece  of  the 
white  matter  of  the  cord,  either  fresh,  or  after  maceration  in  ^ per 
cent,  bichromate  of  potash,  between  a cover-glass  and  a slide. 

(a.)  Observe  the  nerve-fibres,  many  of  them  with  lateral  bulgings, 
or  presenting  a beaded  or  moniliform  appearance.  This  is  due  to 
the  fact  that  these  nerve- fibres  are  devoid  of  a primitive  sheath. 

(6.)  Droplets  of  ‘‘myelin”  with  concentric  markings  are  seen  in 
the  field. 

10.  Nerve-Fibres  of  the  Spinal  Cord  (H). — By  means  of  a 
hypodermic  syringe  make  an  interstitial  injection  of  i per  cent, 
osmic  acid  into  the  white  matter  of  the  antero-lateral  column  of  the 
spinal  cord  of  an  ox.  Tease  a piece  in  glycerine. 

(a.)  Observe  tubes  of  different  sizes,  many  varicose,  with  incisures 
and  cylinder  cones,  but  no  primitive  sheath, 

11.  Staining  the  Cord. — To  stain  sections  the  following  dyes 
may  be  used : — • 

(i.)  Ordinary  carmine,  picro-carmine,  or  acid-carmine.  In  using 
the  last,  use  a very  dilute  (scarcely  coloured)  solution,  and  let  the 
sections  remain  in  the  solution  for  1-2  weeks.  (2.)  These  stains 
may  be  combined  with  haematoxylin.  (3.)  Benzo-azurin.  (4.) 
Aniline-blue  soluble  in  water  but  insoluble  in  spirit.  (5.)  The  same 


342 


PRACTICAL  HISTOLOGY. 


[xxx. 


blue  with  eosin  or  Magdala-red.  If  (5)  be  used  the  axis-cylinders 
are  blue,  the  myelin  rose,  and  there  is  also  a sharp  distinction  in 
colour  between  the  nerve-cells  and  glia-cells.  (6.)  Watery  solution 
of  Congo-red.  The  sections  are  dipped  for  a moment  into  very 
dilute  sulphuric  or  hydrochloric  acid  (i  drop  to  10  cc.  water). 
They  become  blue.  (7.)  Weigert’s  hsematoxylin-copper.  (8.) 
Golgi’s  silver  and  mercuric-chloride  method.  The  Muller’s  fluid 
should  contain  3-3.5  grams  of  potassic  bichromate  to  i gram  of 
sodic  sulphate.  (9.)  Golgi’s  silver  method  and  sections  stained  by 
Magdala-red  {Lavdowsky). 

12.  Tracts  in  the  Spinal  Cord. — These  are  made  out  histo- 
logically by  studying  (a.)  embryonic  cords  in  mammals  from  the  5th 
to  the  9th  month.  These  sections  are  stained  either  by  Weigert’s 
method  (p.  338)  or  by  the  modified  Pal  method.  A T.S.  of  the 
cord  of  a human  foetus  just  before  birth  shows  the  nerve-fibres  in 
the  pyramidal  tracts  still  devoid  of  myelin,  and  thus  they  are  easily 
mapped  out  from  the  other  parts  of  the  cord  which  are  already 
medullatedo 

(Z?.)  The  degeneration  changes  resulting  after  hemi-section  or 
section  of  the  cord.  Section  of  the  cord  is  practised  say  6-10  days 
before  the  cord  is  required.  Parts  of  the  cord  above  and  below’  the 
seat  of  injury  are  hardened  for  10  days  or  so  in  Muller’s  fluid. 
Thin  pieces  are  then  placed  for  several  days  in  Marchi’s  fluid 

(P.  347). 

Sections  are  mounted  in  balsam.  All  the  nerve-fibres  which 
have  undergone  degeneration  are  stained  black ; healthy  nerve- 
fibres  are  yellowish  (p.  347).  Or  instead,  Weigert’s  process  may  be 
used,  as  then  the  degenerated  tracts  remain  unstained  owing  to  the 
absence  of  myelin. 


ADDITIOIs^AL  EXEECISES. 

13.  Dry  Preparation. — Stain  a T.So  with  methylene-blue  (i  per  cent). 
Wash  it,  and  allow  it  to  dry  on  a slide.  Add  a drop  of  balsam.  This  shows 
very  well  the  general  characters  of  the  cord  ; the  multipolar  nerve-cells  are 
somewhat  shrunken,  but  still  they  and  their  processes  are  well  stained. 

14.  Sections  in  Ehrlich-Biondi’s  Fluid. — Place  sections  in  this  fluid  (p,  81) 
well  diluted  (i  : 40),  and  heat  them  in  a watch-glass  until  vapour  is  just  given 
off.  Mount  them  in  balsam,  clarifying  either  with  xylol  or  aniline-oil  and 
xylol  (Lesson  III.  16).  The  glia-flbrils  are  violet  in  tint,  and  so  is  the  con- 
nective tissue  generally  ; the  nuclei  of  the  glia-cells  bluish,  the  myelin  orange, 
the  axis-cylinder  somewhat  violet.  The  cells  in  the  grey  matter  have  a 
pleasant  violet  tint.  Benzo-azurin  can  be  used  in  the  same  way. 

15.  T.S.  Cord.  Eosin  and  Logwood. — The  neuroglia-cells,  connective- 
tissue,  and  epithelium  of  the  central  canal  have  a logwood  tint,  the  other  parts 
are  rosy. 


XXX.]  SPINAL  CORD.  343 

16.  Staining  the  Cord  in  Bulk  in  Aniline  Blue-Black. — Small  pieces  of  the 
cord  are  placed  in  the  following  fluid  for  a day  or  two  : — 


Aniline  blue-black 

. . 2 grams, 

Water. 

. . 60  cc. 

Alcohol 

. 40  „ 

Mount  the  sections  in  balsam. 

17.  Transverse  Markings  on  Axis-Cylinders  and  Nerve-Cells. — Place  small 
pieces  of  a perfectly  fresh  cord  in  i per  cent,  silver  nitrate  solution,  and  keep 
them  in  the  dark  for  forty-eight  hours,  renewing  the  fluid  several  times. 
Wash  the  pieces  and  place  them,  exposed  to  light,  in  the  following  mixture  : — 
Formic  acid(i  part),  amylic  alcohol  (i  part),  and  water  (100  parts),  for  5-7 
days.  Tease  a fragment  in  glycerine  and  observe  the  alternate  brown  and 
clear  markings  on  the  axis-cylinders  (Frommann’s  lines)  and  on  the  nerve-cells 
(Jakimovitch)A 

18.  Isolated  Neuroglia-Cells. — (a.)  These  are  obtained  by  the  interstitial 
injection  of  osmic  acid  into  the  white  matter  of  the  cord  (Lesson  XXX.  10.) 
A small  piece  is  teased  and  stained  with  picro-carmine. 

(H)  Observe  the  branched  cell,  with  a granu- 
lar body  and  long  processes  (fig.  321).  It  re- 
quires considerable  care  to  dissociate  such  a 
cell,  and  it  must  usually  be  looked  for  and 
isolated  with  the  aid  of  a dissecting  microsco|  e 
(p.  22). 

{b.)  In  sections  of  the  cord  prepared  by 
Golgi’s  method,  neuroglia-cells  may  be  stained 
along  with  the  nerve-cells,  and  on  other  occa- 
sions they  may  be  the  only  elements  on  which 
the  silver  or  mercury  takes  effect.  Note  that 
each  cell  gives  off  many  very  fine  processes. 

Some  of  the  latter  may  be  seen  to  become 
attached  to  the  walls  of  a capillary  or  other 
blood-vessel. 

(c.)  Isolated  glia-cells  may  be  found  after 
maceration  in  Landois’  fluid  (p.  26),  and  subsequent  staining  with  Magdala- 
red. 

19.  Isolated  Nerve-Cells  of  the  Cord. — The  best  dissociating  reagents  are 
dilute  alcohol  or  Landois*  fluid  (3-4  days,  p.  26).  A staining  fluid  may  be 
added  to  the  dilute  alcohol,  and  thus  dissociation  and  staining  go  on  simul- 
taneously, With  cells — either  nerve  or  glia — isolated  by  Landois’  fluid,  it  is 
better  to  stain  after  maceration.  The  best  stains  are  Magdala-red,  methyl- 
blue  II.  (0.5-1  per  cent.,  5-10,  drops  added  to  10  cc.  of  the  macerating  fluid). 
Lavdowsky^  uses  a “semidesiccation  method ’*  like  that  used  for  connective 
tissue ; the  isolated  cells  are  allowed  to  become  nearly  dry,  and  then  alcohol 
is  slowly  added  to  remove  the  remainder  of  the  water  (see  also  Lesson  XVIII. ). 
Or  after  maceration  shake  the  tissue  in  a very  small  quantity  of  water  in  a test- 
tube,  and  place  it  in  a watch-glass,  add  6-10  drops  of  glycerine  and  a little 
picro-carmine,  and  dry  the  whole  over  a sulphuric  acid  desiccator.  This 
removes  all  the  water,  and  one  has  then  the  cells  stained  in  glycerine  ready  to 
be  mounted. 

20.  Pal’s  Method  of  Staining  Nerve-Fibres.  — This  method  requires  consider- 
able care.  Harden  the  cord  or  brain  in  Muller’s  fluid,  make  T.S.,  and  place 
them  in  alcohol.  Stain  in  .75  per  cent,  watery  solution  of  hsematoxylin  con- 

1 Journ.  de  V Anat.  et  de  la  Phys.,  xxiv.  p.  142,  1888. 

2 Archivf,  mik,  Anat.^  xxxviii.  p.  264. 


Fig.  321.— Isolated  Neuroglia-Cell 
of  Spinal  Cord  of  Ox.  n.  Nu- 
cleus ; c.  Granular  proto- 
plasm ; /.  Fibres  of  neuroglia. 


344 


PRACTICAL  HISTOLOGY. 


[xxx. 

taining  some  alcohol  and  a few  drops  of  a saturated  solution  of  lithium 
carbonate  (6-io  hours).  They  are  then  “differentiated”  in  J per  cent,  solu- 
tion of  permanganate  of  potash  (10-15  seconds),  and  subsequently  in  the 
following  mixture : — 

Oxalic  acid  . , , i gram. 

Potassic  sulphite  , . i ,, 

Distilled  water  , , . 200  cc. 

When  the  grey  and  white  matter  are  differentiated,  the  sections  maybe  stained 
in  carmine,  safranin,  or  alum-carmine,  and  mounted  in  balsam. 

Stages  of  PaPs  Methode 
(1.)  Harden  in  Muller’s  fluid. 

(2.)  Cut  sections  and  stain  in  Weigert’s  hsematoxylin  (24-48  hours). 

(3.)  Wash  in  water  to  which  1-2  per  cent,  lithium  carbonate  is  added.  Tim 
sections  must  be  deep  blue. 

(4.)  Differentiate  the  sections  in  0.25  per  cent,  potassic  permanganate  solu- 
tion (20-30  secs. ) till  grey  matter  becomes  yellow. 

(5.)  Transfer  to  oxalic  acid  solution  (few  secs.). 

(6.)  Wash  in  water,  and  dehydrate  in  alcohol,  xylol,  balsam. 

This  method  can  be  done  rapidly.  Only  the  nerve-fibres  are  stained  ; the 
intervening  parts  may  be  stained  subsequently  with  picro-carmine. 

21.  Vessale’s  Modification  of  Weigert’s  Method.  ^ 

(1.)  Harden  in  Muller’s  fluid  and  afterwards  in  alcohol. 

(2.)  Stain  celloidin  sections  (3-5  mins.)  in  i per  cent,  hfematoxylin 
dissolved  in  warm  water.  Allow  it  to  cool.  Tlie  sections 
become  black. 

(3. ) Place  in  saturated  filtered  solution  of  neutral  copper  acetate  (3-5 
mins.)  and  then  lave  in  water. 

(4.)  Differentiate  in  a solution — 

Borax  ...  2 grams. 

Potassic  ferridcyanide  , 2.5  ,, 

Water  , . , 300  cc. 

Ganglion-cells,  glia-cells,  and  degenerated  parts  soon  become  decolorised, 
only  the  medullated  fibres  remain  dark-vioiet. 

(5.)  Wash  thoroughly  in  water.  They  may  be  stained  wdth  alum- 
carmine  as  a contrast  stain. 

(60)  Absolute  alcohol.  Xylol-carbolic  acid  (3  : i),  remove  surplus  with 
bibulous  paper.  Balsam. 

22.  Kulschitzky’s  Method  for  Medullated  Fibres.  2 

(1.)  Harden  in  Muller’s  or  Erlicki’s  fluid  (1-2  mins,),  then  wash  in 
water  (1-2  days).  Alcohol.  Make  sections  in  celloidin. 

(2.)  Stain  (1-3  hours)  in  his  acid  hsematoxylin  (p.  342). 

(3.)  Difterentiate  (2-3  hours)  in 

Sat.  sol.  lithium  carb.  , . loo  cc. 

Ferridcyanide  of  potash  (i  per  cent.)  10  ,, 

(4.)  Wash  thoroughly  in  water.  Balsam. 

23.  Golgi’s  Silver  Methods^ — (a.)  Slow  Method. — Small  parts  of  the  cord  or 

^ Zeit,  wiss.  Mikros.^  vii.  p.  517,  and  Archiv,  ital,  de  Biol.,  xv.  p.  158, 
1891. 

2 Anat.  Anzeig.,  iv.  p.  519,  1890. 

^ Sulla  Jina  anatomiadegli  organi  centrali  del  sistema  nervoso,  Milano,  1886. 


XXX.] 


SPINAL  CORD. 


345 


brain,  after  being  hardened  in  2 per  eent.  potassium  bichromate  for  3-4  days, 
are  then  translerred  to  a stronger  solution,  say  3 per  cent.,  for  4 days.  In- 
crease successively  the  quantity  of  the  bichromate  until  4-6  per  cent,  is 
reached.  It  takes  30-50  days,  according  to  temperature  or  other  circum- 
stances, to  get  the  tissues  properly  hardened.  The  hardened  parts  are  then 
transferred  for  24-48  hours  to  .5-.  75  per  cent,  silver  nitrate.  It  is  better  to 
place  them  first  of  all  in  a small  quantity  of  silver  nitrate,  and  to  wash  them 
in  this  fluid.  The  fluid  becomes  of  a dark  orange  tint  from  silver  chromate. 
Place  the  pieces  in  fresh  silver  nitrate.  Harden  in  alcohol.  Make  hand 
sections,  clarify  them  in  the  usual  way,  and  mount  in  balsam,  but  donotapj)ly 
a cover-glass. 

(b.)  Bapid  Method. — Make  the  following  mixture  : — ■ 

Osmico- Bichromate  Mixture. 

Potassic  bichromate  (3  per  cent.)  , . 20  cc. 

Osmic  acid  (i  per  cent.)  , . . 5 ,, 

Place  small  pieces  of  the  fresh  organ  in  15  cc.  of  this  fluid  for  2-3  days,  and 

then  place  them  in  the  silver  solution  (.5-1  per  cent.;  for  1-2  days.  I have 
found  the  cells  of  the  cerebral  cortex  stained  black  two  or  three  hours  after 
immersion  of  the  organ  in  silver  nitrate. 

This  is  an  excellent  method,  specially  useful  for  young  or  embryonic  nervous 
system,  as  the  fluid  penetrates  more  readily  where  tlie  myelin  is  scanty.  It  is 
very  capricious  in  its  action.  Sometimes  only  small  parts  are  stained.  The 
nerve-cells  and  their  processes,  and  nerve-fibres  without  myelin,  and  axis 
cylinders  are  stained  black. 

(c.)  Medium  Method. — Small  pieces  of  the  fresh  tissue  are  placed  for  3-5  days 
in  3 per  cent,  potassic  bichromate ; then  3-4  days  in  the  osmico-bichromate 
mixture  as  above,  and  then  in  silver  nitrate. 

It  is  not  a matter  of  indifference  which  method  is  used.  Golgi’s  method 
only  stains  certain  elements — nerve-cells,  neuroglia-cells,  and  sometimes 
blood-vessels.  Thus,  by  the  rapid  method,  in  the  case  of  the  cerebellum,  the 
parallel  fibres  and  the  granular  layer  are  the  chief  parts  stained;  Purkinje’s 
cells  stain  best  by  the  slow  method.  As  a general  rule,  the  axis  cylinders  and 
their  processes  stain  best  by  means  of  the  rapid  and  medium  methods,  the 
slow  method,  as  a rule,  staining  best  the  protoplasmic  processes. 

The  rapid  silver  method  has  been  fextensively  used  by  Golgi  and  his  ]>upils, 
by  Kolliker,  but  above  all  by  Ramon  y Cayal.  The  black  deposit  Golgi  calls 
a “ black  reaction.  ” 

24.  Golgi  and  Mondino’s  Sublimate  Method. — Small  pieces  of  the  central 
nervous  system,  after  hardening  in  bichromate  of  potash,  are  placed  in  a 
watery  solution  (.25  per  cent.)  of  corrosive  sublimate.  The  volume  of  fluid 
must  be  large,  and  renewed  frequently,  ^.e.,  as  often  as  it  is  yellow.  After 
10-15  days  the  reaction  has  occurred  in  small  pieces  ; but  it  is  better  to  expose 
the  })ieces  longer,  than  this  to  the  action  of  the  salt ; in  fact,  prolonged  im- 
mersion rather  improves  it.  The  tissues  may  remain  for  months  in  the  fluid 
without  disadvantage.  The  sections  must  be  very  thoroughly  washed,  else, 
after  being  mounted,  needle-shaped  crystals  of  the  sublimate  are  apt  to  form. 
The  sections  are  mounted  in  balsam  or  glycerin  ; only  they  are  mounted  with- 
out a cover-glass. 

In  Golgi’s  silver  method  the  cells,  and  in  some  preparations  the  blood- 
vessels as  well,  are  opaque  and  black.  Sometimes  the  body  of  the  cell  and  its 
finest  ramified  processes  can  be  seen  with  the  utmost  sharpness.  The  silver  is 
deposited  only  on  the  cells  and  their  processes,  not  on  the  nerve-fibres.  The 
sections,  however,  are  often  dotted  over  with  a black  metallic  deposit.  In  the 
mercury  preparations,  if  the  cells  do  not  appear  to  be  very  black,  they  may  be 
darkened  by  washing  them  in  sodic  sulphite. 

31 


346 


PRACTICAL  HISTOLOGY. 


[xxx. 


The  sublimate  may  act  on  {a.)  the  ganglionic  cells,  (5.)  the  neuroglia  cells, 
and  (c. ) the  blood-vessels.  The  best  results  with  these  methods  are  obtained 
with  the  cerebral  cortex.  The  sections  can  be  stained  afterwards  by  the  usual 
methods,  and  particularly  by  Magdala-red. 

The  parts  acted  on  by  the  sublimate  are  white  by  reflected  light,  and  appear 
black  by  transmitted  light,  because  they  are  opaque.  Golgi ^ finds  that  for  the 
study  of  the  diffuse  network  in  the  central  nervous  system,  by  this  method  the 
best  results  are  obtained  when  the  “metallic  white’*  is  converted  into  a 
metallic  black.  For  this  purpose  he  uses  the  “fixer”  employed  for  fixing 
positive  photographs  on  aristotypic  paper. 

(A.)  Water.  , 

Hyposulphite  of  soda  , 

Alum  .... 

Ammonium  sulphocyanide 
Chloride  of  sodium  » 

Leave  the  mixture  for  8 days  and  then  filter. 

(B.)  Water,  , • 

Gold  chloride. 

For  the  toning  and  fixing  fluid,  mix  of 

A . . . . 

B . . . . 

Procedure. 

(1.)  Wash  in  distilled  water. 

(2.)  Immerse  sections  (i-2  mins.)  in  the  above  mixture.  The  sections 
become  black. 

(3.)  Prolonged  washing  in  water. 

(4.)  (Optional.)  Faint  coloration  of  the  sections  with  acid  carmine. 

(5.)  Wash  again,  then  mount  in  balsam. 

25.  Double  Impregnation  Method  of  Bamon  y Cayal.^ — Morsels  of  tissue 
are  placed  in  the  dark  in  the  osmico-bichromate  mixture  as  for  Golgi’s  rapid 
method  (2-3  days),  and  then  they  are  gently  washed  and  placed  in  .5-.  75 
per  cent,  silver  nitrate  (1-2  days).  They  are  retransferred  for  3-4  days  to  the 
osmico-bichromate  fluid  (3-4-5  days),  and  then  again  to  silver.  They  are  then 
hardened  in  alcohol  and  cut.  This  method  certainly  giv^es  good  results  in 
some  cases,  and  is  very  useful  for  the  brain  and  embryo  preparations. 

26.  Embryo  Cords. — (a.)  Harden  the  spinal  cord  of  an  embryo  chick  at  the 
9th  day  as  above.  Remove  as  much  as  possible  of  the  surrounding  vertebral 
column  before  placing  it  in  the  osmico-bichromate  mixture. 

(L)  Observe  the  axis-cylinders  of  the  fibres  of  the  anterior  roots  springing 
from  the  axis-cylinder  processes  of  the  multipolar  branch  cells  of  the  anterior 
cornu,  themselves  black.  Note  that  the  fibres  of  the  posterior  root  enter  the 
cord  and  sj)lit  up  into  fibrils. 

(b.)  Collateral  Fibres. — Harden  the  spinal  cord  of  an  embryo  mammal  {e.g., 
sheep,  20-25  cm.  long)  or  embryo  of  chick  in  the  above  fluid,  and  make  L.S. 
in  the  line  of  entrance  of  the  posterior  roots.  The  sections  need  not  be  very 
thin,  but  one  may  have  to  make  several  preparations  before  one  gets  a satisfac- 
tory result. 

(H)  Note  that  when  a fibre  of  the  posterior  root  enters  the  white  matter 
of  the  cord  it  divides,  sending  one  branch  upwards  and  one  downwards  (fig. 
322),  which  run  for  a distance  more  or  less  longitudinally,  but  many  enter  the 

^ Archiv.  ital.  de  Biologie,  xv.  p.  462,  1891. 

* Internat.  Monatsch,  f,  Aumt,  u,  Phys.^  vi.  p.  170. 


I litre. 
175  grams. 
20'  „ 

10  „ 

40  » 

100  cc. 

I gram. 

60  cc. 

7 „ 


SPINAL  CORD. 


XXX.] 


347 


grey  matter,  and  end  free  in  fine  bra; 
nerve-cells.  The  fibres  give  off  at 
right  angles  to  their  course  fine  fibrils 
— collaterals— which  enter  the  grey 
matter,  divide  into  fibrils,  and  end 
free. 

27.  Degeneration  of  the  Cord — 
(a.)  Method  of  Marchi  for  degenera- 
tion, in  central  nervous  system  or  in 
nerves  (Lesson  XVll.)  ; — 

(1.)  Harden  very  small  pieces  of  a 
nerve  or  spinal  cord — either  of 
which  is  undergoingdegenera- 
tion — in  Miiller’s  fluid  for  at 
least  8 days. 

(2.)  Place  for  6 days  in  the  follow- 
ing 

Muller’s  fluid  • • 2 parts. 

Osmic  acid  (i  per  cent.)  i part. 

(3.)  Wash  in  water,  harden  in 
alcohol. 

(4.)  Embed  and  cut  in  celloidin. 

All  degenerated  parts  appear  black, 
all  the  others  light  grey  or  yellowish 
(see  p.  212). 

(&.)  Weigert’shsematoxylin method 
may  be  used  for  the  same  purpose. 
The  degenerated  fibres  are  unstained, 
i.e.y  on  differentiating  the  section 
the  degenerated  parts  rapidly  give  up 


without  forming  connections  with 


t'lG.  322.— L.S.  of  the  Cord  of  the  Cervical 
Region  of  an  Embryo  Sheep  (22  cm.  long), 
to  show  division  of  posterior  roots  after 
entering  the  spinal  cord. 

heir  stain,  and  thus  appear  unstained. 


LESSOR  XXXI. 

MEDULLA  OBLONGATA— CEREBELLUM  - 
CEREBRUM. 

MEDULLA  OBLONGATA  OR  BULB. 

The  medulla  oblongata  is  hardened  in  the  same  way  as  the  cord. 
T.S.  are  made  at  different  levels,  and  stained  in  the  same  way 
as  the  cord  (L  and  H). 

1.  T.S.  Decussation  of  Pyramids. 


348 


PRACTICAL  HISTOLOGY. 


[XXXI. 


(a.)  Observe  the  shape  of  the  cord,  the  decussation  of  the  anterior 
pyramids,  z.e.,  bundles  of  fibres  are  seen  coming  from  the  lateral 
column  of  one  side,  and  running  inwards  and  towards  the  middle 


line,  , thus  separating 


Fig.  323.— T.S.  Medulla  Oblongata  through 
Decussation  of  Pyramids.  D.Py.  An- 
terior pyramid  ; Fa.  Anterior  Cornu ; 
Ng.  Nucleus  of  the  funiculus  gracilis  ; 
g.  Substantia  gelatinosa;  XI,  Spinal 
accessory  nerve. 


somewhat  the  anterior  from  the  posterior 
cornua  (fig.  323).  They  actually 
pass  to  the  anterior  pyramid  of 
the  opposite  side  of  the  medulla. 

{h.)  At  the  anterior  part,  on 
each  side  of  the  anterior  median 
fissure,  sections  of  the  anterior 
pyramid. 

{r.)  At  the  posterior  part,  a 
small  part  of  the  posterior  cornu 
extending  backwards  as  the 
nucleus  of  the  clava  in  the 
funiculus  gracilis.  Another  mass 
of  grey  matter  in  the  funiculus 
cuneatus. 

2.  T.S.  of  Olivary  Bodies  (L 

and  H).  — Observe  the  folded  mass 
of  grey  matter,  with  many  multipolar  nerve-cells,  constituting  the 
olivary  nucleus  (fig.  324);  the  complex  of  fibres,  horizontal,  vertical, 

and  those  cut  longitudinally, 
constituting  the  formatio 
reticularis ; the  much  altered 
arrangement  of  the  grey 
matter,  which  appears  in  the 
floor  of  the  fourth  ventricle  ; 
and,  according  to  the  level 
at  which  the  section  is  made, 
there  may  be  met  with  the 
origin  of  certain  of  the  cranial 
nerves  (fig.  324). 

Of  course  sections  should 
be  made  from  higher  levels 
in  the  medulla,  and  also 
through  the  pons.  If  a student  has  the  requisite  time,  it  is  best  to 
make  a series  of  sections  from  below  upwards,  fixing  them  in  order 
on  slides. 

The  two  following  tables  show,  the  one  the  fissures,  areas,  and 
mouldings  to  be  noted  on  the  medulla  oblongata,  and  the  other  the 
grey  matter  of  the  cord  and  meduUa  oblongata. 


Pig.  324. — T.S.  Medulla  Oblongata  at  the  Level 
of  the  Olivary  Body  ; partly  Diagrammatic. 


XXXI.] 


MEDULLA  OBLONGATA. 


349 


Objects  seen  on  the  Surface  of  the  Medulla  Oblongata. 

C Anterior  median. 

A.  Fissures.  -!  Posterior  median. 

l^Two  lateral. 


B.  Areas. 


I.  Anterior  area 
{between  A . M.  F,  and 
Olive). 


I Anterior  pyramids. 

I Decussation  of  pyramids. 


2.  Lateral  area  ] olivary  body. 
{between  roots  of  lUh  V 
and  1 2th  nerves).  J 


3.  Posterior 
area. 


C.  External  Arciform  Fibres. 


^ f Restiform  body. 

Lower  ) Funiculus  of  Rolando  and  its  tubercle. 
Part.  1 Funiculus  cuneatus  and  its  tubercle. 

( Funiculus  gracilis  and  its  clava. 

^art  ^ j-  Floor  of  fourth  ventricle. 


Table  of  Grey  Matter  of  the  Medulla  Oblongata. 


Grey  Matter 
of  the 

Spinal  Cord. 


Cord. 

Anterior  j gead 
I Base 

f Head 
Posterior  | 

I Base 


Medulla. 

...  Nucleus  lateralis. 

...  Anterior  part  of  formatio  reticularis. 

..  Nucleus  of  the  fasciculus  teres. 

...  Nucleus  of  Rolando. 

...  Posterior  part  of  formatio  reticularis. 
f Nucleus  of  the  funiculus  gracilis  (clava). 
Nucleus  cuneatus. 

(^Nucleus  on  floor  of  fourth  ventricle. 


Isolated  Grey  Nuclei/ 

A f»pp«enr\r  n ivnrv  tiiip  pi 


in  the  Medulla. 


Accessory  olivary  nuclei. 

Nucleus  of  external  arciform  fibres. 


CEREBELLUM. 

Methods. — The  cerebellum  is  hardened  in  the  same  way  as  the 
cord.  It  is  advantageous,  however,  to  wash  out  the  blood-vessels 
of  the  whole  brain  with  Muller’s  fluid,  and  afterwards  to  distend 
them  with  the  same  fluid. 

If  ammonium  bichromate  be  used  (2  percent.),  the  pieces  harden 
quicker  than  in  the  case  of  the  cord. 

For  the  application  of  Golgi’s  method,  see  p.  345. 

3.  V.S.  Cerebellum,  z.e.,  including  the  grey  and  white  matte*’ 


350 


PRACTICAL  HISTOLOGY. 


[XXXI. 


cut  at  right  angles  to  the  direction  of  the  folds.  Stain  it  with 
carmine,  aniline  blue-black,  or,  better  still,  first  wuth  logwood  and 
then  with  eosin.  Mount  in  balsam.  The  best  way  for  the  student 
is  to  stain  a small  piece  of  the  cerebellum  in  hulk  in  borax- 
carmine  for  two  or  three  days,  or  longer,  and  then  embed  and 
cut  in  paraffin.  In  this  way  there  is  no  fear  of  the  sections  break- 
ing up,  and  the  relative  positions  of  the  several  parts  are  accurately 
maintained. 

In  making  sections  of  the  cerebellum,  it  is  important,  if  one 
wishes  to  see  the  wide  expanse  of  the  protoplasmic  processes  of 
Purkinje’s  cells,  to  make  sections  across  the  direction  of  the  laminae. 
If  made  in  the  direction  of  the  laminae  the  leash  of  protoplasmic 
processes  appear  quite  narrow”  (fig.  330,  B).  The  protoplasmic 
processes,  or  dendrites,  as  they  have  been  called  by  His,  spread  out 
in  planes  transverse  to  the  direction  of  the  lamellae,  hence  the 
necessity  for  the  above  precaution. 

Suppose  a haematoxylin-eosin  specimen  to  be  prepared. 

(a.)  (L)  Observe  the  primary  and  secondary  convolutions  (fig. 
325).  In  each  leaflet  from  within  outwards,  the  white  matter, 


composed  of  medullated  nerve-fibres,  and  outside  this  the  grey 
matter,  composed  of  two  layers,  viz. : — (i.)  The  nuclear  layer, 
composed  of  many  layers  of  small  nuclei — stained  blue — each 
surrounded  by  a very  small  quantity  of  protoplasm.  (ii.)  The 
outer  layer  of  the  cortex,  thicker  than  (i.),  with  a somewhat 
granular  appearance,  and  containing  branches  of  Purkinje’s  cells 
and  some  small  branched  angular  nucleated  cells  (fig.  326). 


Human  Cerebellum, 
X 10. 


Fig.  326. — Cortex  of  the  Cerebellum,  x 90. 
a.  Outer;  h.  Inner  or  granular  layer; 
p.  Cells  of  Purkinje. 


CEREBELLUM. 


XXXI.] 


351 


Numerous  blood-vessels  enter  the  surface  of  the  cerebellum  from 
the  pia  mater  covering  it. 

(b.)  At  the  boundary-line  between  (i.)  and  (ii.)  a row  of  large 
cells — Purkinje’s  cells — each  with  a somewhat  oval  or  globular 
body,  with  a single  central  process,  which  becomes  continuous  with 
a nerve-fibre,  although  this  is  not  seen  in  this  preparation.  Each 
cell  gives  off  a peripheral  process,  which  immediately  branches,  the 
larger  branches  running  laterally  for  a short  distance,  and  each 
branch  divides  again  and  again,  the  fine  branches — protoplasmic 
processes  or  dendrites — running  vertically  through  the  outer  layer 
of  the  cortex  nearly  to  its  free  surface.  The  branched  arrangement 
of  these  fibres  has  been  compared  to  the  antlers  of  a stag. 

By  means  of  the  rapid  Golgi  method,  a basket-shaped  complex 
of  fibrils  can  be  seen  round  the  basis  of  Purkinje’s  cells. 

(c.)  (H)  The  granular  or  nuclear  layer,  with  two  kinds  of  nuclei 
or  rather  cells.  The  most  numerous  are  small  and  granular,  and 
arranged  in  groups ; they  are  stained  violet  by  the  hsematoxylin. 
The  others  (larger  and  spherical,  with  a nucleolus)  are  the  nuclei 
of  small  ganglionic  nerve-cells.  They  are  stained  reddish.  Amongst 
the  granules  may  be  seen  medullated  fibres  stained  reddish. 

The  more  recent  methods  of  Golgi  and  Bam6n  y Cayal  show 
that  these  two  kinds  of  cells  correspond  to  nerve-cells.  The 
small  cells  have  an  axis-cylinder  and  several  protoplasmic  pro- 
cesses, while  the  large  cells  in  some  respects  resemble  small 
Purkinje’s  cells. 

4.  Blood-Vessels  of  the  Cerebellum. — These  are  injected  when 
the  blood-vessels  of  an  animal  are  injected  from  the  aorta  with  a 
Berlin-blue  or  carmine-gelatine  mass.  Mount  an  unstained  sectin 
in  balsam  ( L and  H). 

(a.)  Observe  the  vascular  pia  mater  sending  at  intervals  long  or 
medullary  branches  through  the  cortex  to  the  medulla,  and  short 
or  cortical  branches  which  break  up  into  a rich  plexus  of  capil- 
laries in  the  cortex,  so  that  the  latter  is  far  more  vascular  than  the 
white  matter.  Each  vessel  is  surrounded  by  a perivascular  lymph- 
space. 

CEREBRUM. 

Methods. — Harden  it  in  the  same  way  as  the  cord  and  cerebellum. 
For  Golgi’s  methods  (p.  345). 

If  Weigert’s  method  is  used,  the  sections  must  remain  for  twenty- 
four  hours  in  the  hsematoxylin  solution  in  order  to  see  the  plexus  of 
medullated  fibres  in  the  superficial  layers  of  the  cortex,  and  three 
hours  for  the  fibres  which  ascend  between  the  pyramidal  cells  of 
the  cortex  cerebri. 

5,  V.S.  Cerebrum. — Make  Y.S.  through  the  central  part  of  the 


352 


PRACTICAL  HISTOLOGY. 


[XXXI. 


brain  of  a small  animal.  In  the  case  of  a human  brain,  select  the 
ascending  parietal  or  ascending  frontal  convolution.  Make  V.S. 
either  by  freezing  or  in  paraffin  after  staining  in  bulk  in  borax- 
carmine.  Other  sections  may  be  stained  in  aniline  blue-black,  and 
all  are  mounted  with  balsam. 

With  the  naked  eye  observe  the  shape  of  the  convolution,  the 
grey  matter  outside,  of  a certain  thickness  and 

deeply  stained,  and  inside  it  the  white  matter  less  deeply  stained. 

(L  and  H)  («.)  Observe  the  white  matter,  composed  of  medul- 
lated  fibres,  with  leucocytes  here  and  there  between  them. 

(/;.)  Outside  this  the  grey  matter,  composed  of  several  layers, 
recognised  by  the  arrangement  and  shape  of  the  cells  present  in  it. 


It  will  depend  very  greatly  upon  the  plane  of  the  section  whether 
the  student  sees  all  the  layers  in  any  single  section.  They  are 
usually  in  a five-layer  type,  but  the  relative  thickness  of  the  layers 
varies  in  different  parts  of  the  cerebrum. 

A.  Arrangement  of  Nerve-Cells, 

(c.)  The  layers  from  the  surface  inwards  are  : — 

(i.)  The  narrow  outer  or  first  layer  (or  finely-granular  or  mole- 
cular layer)  consists  of  a network  of  fibrils  with  a very  few  small 
cells.  Chiefly  neuroglia  cells,  mostly  vertical  to  the  surface. 

By  Golgi’s  method,  it  can  be  shown  to  contain  a layer  of  medul- 
lated  fibres  just  under  and  parallel  to  the  pia,  and  also  some 
branched  non-medullated  fibres.  It  also  contains  a few  small 
nerve-cells  with  two  or  more  axis-cylinder  processes.  The  latter 
details  can  only  be  detected  in  a preparation  made  by  Golgi’s 


Fig. 

Ascending  Frontal  Convolution, 
i.e.,  a Motor  Area.  Carmine, 
X 20. 


Fig.  328.— V.S.  Middle  Frontal 
Convolution.  Carmine,  x 20. 


XXXI.]  CEREBRUM.  353 

method.  In  it  may  be  seen  sections  of  blood-vessels  passing  from 
the  pia. 

(ii.)  The  second  layei\  or  layer  of  small  pyramidal  cells,  is  usually 
narrow,  with  several  rows  of  small  pyramidal  cells,  the  peripheral 
processes  of  the  latter  pointing  towards  the  surface  of  the  convolu- 
tion. It  passes  gradually  into 

(iii.)  The  third  layer ^ or  layer  of  large  pyramidal  cells,  which  is 
much  thicker  than  the  others,  and  contains  large  pyramidal  cells, 
each  with  a peripheral  process,  which  can  sometimes  be  traced 
outwards  for  a considerable  distance.  The  axis-cylinder  process 
gives  off  several  collaterals,  and  can  be  traced  into  a medullated 
fibre  of  the  white  matter.  The  cells,  however,  may  be  cut  obliquely, 
and  then  they  appear  triangular.  Usually  the  cells  are  larger  in 
the  deeper  layers,  and  become  smaller  in  the  outer  layers. 

(iv.)  The  fourth  layer,  or  layer  of  irregular  or  polymorphous  cells. 
This  is  a narrow  layer  of  small,  usually  angular  but  irregularly- 
shaped  cells.  They  lie  between  the  nerve-fibres  which  pass  into 
the  cortex.  In  the  motor  areas  of  the  frontal  and  parietal  convolu- 
tions large  pyramids  arranged  in  groups  or  nests  may  be  found 
between  these  cells. 

(v.)  The  fftli  layer,  or  layer  of  fusiform  cells,  also  a narrow  layer, 
with  a few  fusiform  cells  with  nerve-fibres  between  them.  This 
layer  abuts  on  the  white  fibres  of  the  medulla,  and  may  in  some 
regions  be  fused  with  the  previous  layer.  In  the  grey  matter 
superficial  to  the  island  of  Reil,  it  exists  as  a separate  layer  consti- 
tuting the  claustrum. 

Xot  unfrequently  in  bichromate  and  chromic  acid  preparations, 
the  place  of  the  pyramidal  cells  is  partly  represented  by  clear 
spaces,  produced  by  vacuolation  of  the  cells.  It  is  by  no  means 
easy  to  distinguish  a nerve-cell  from  a neuroglia-cell  in  the  cere- 
brum. Usually  the  glia-cell  nuclei  are  smaller.  Between  the  rows 
of  cells  may  be  seen  fine  longitudinal  striation,  indicating  the  exist- 
ence of  nerve-fibres  in  the  grey  matter. 

The  structure  of  the  cortex  is  not  identical,  although  similar, 
throughout.  In  the  motor  areas  especially,  i.e.,  in  the  ascending 
frontal,  ascending  parietal,  and  part  of  the  marginal  convolutions, 
the  pyramidal  cells  are  usually  larger  than  in  those  areas  which  are 
described  as  sensory — e.g.,  the  occipital,  temporo-sphenoidal,  or  even 
in  the  anterior  part  of  the  frontal. 

It  is  advisable,  therefore,  to  provide  the  student  with  sections 
from  these  different  areas.  There  is  no  abrupt  transition  between 
one  type  of  cortical  structure  and  another. 

B.  Course  of  White  Fibres  in  the  Cortex  Cerebri. — This  cannot 
be  made  out  in  sections  prepared  as  above.  All  that  can  be  seen  is 
that  fine  strands — radii — of  medullated  fibres  pass  from  the  white 

z 


354 


PRACTICAL  HISTOLOGY. 


[XXXI. 


centre  at  intervals  into  the  grey  matter,  and  run  outwards  as 
medullary  rays  between  the  nerve-cells.  By  other  methods  it  can 
he  shown  that  some  of  them  become  continuous  with  axis-cylinder 
processes  of  the  pyramidal  and  polymorphous  cells,  while,  as  shown 
in  fig.  331,  others  end  in  free  arborisations.  Tracing  the  fibres  the 
other  way,  i,e.^  as  axial-cylinder  processes  of  pyramidal  cells,  it  has 
been  shown  by  other  methods  that  some  of  them — chiefly  from  the 
large  pyramids  of  the  motor  areas — pass  into  the  white  matter, 
enter  the  corona  radiata,  and  pass  through  the  inner  and  anterior 
two-thirds  of  the  posterior  division  of  the  internal  capsule  to  enter 
and  form  the  pyramidal  tracts.  They  constitute  the  projection 
fibres,  and  end  in  free  arborisations  in  relation  with  the  multi- 
polar nerve-cells  of  the  anterior  cornu  of  the  grey  matter  of  the 
cord. 

The  so-called  commissural  or  callosal  fibres,  which  join  opposite 
halves  of  the  brain,  pass  into  the  corpus  callosum  either  directly  or 
by  collateral  fibres  (fig.  331)  and  pass  to  the  opposite  side  to  end  as 


Fig.  329. — Injected  Cerebral  Cortex  of  Dog.  1.  Layer  with  few  vessels ; 2.  Layer  of  large 
pyramidal  cells  ; 3.  Deepest  layers  of  cortex  ; 4.  Medulla. 

free  arborisations  in  the  grey  matter  there  (fig.  331,  /).  Others  are 
said  to  join  the  antero-posterior  fibres  (fig.  331,  B)  or  association 
fibres,  whicli  run  between  grey  matter  in  different  parts  of  the  same 
hemisphere. 

6.  V.S.  Middle  Frontal  Convolution  (fig.  328). — Compare  this 
with  that  from  a motor  area,  and  note  the  absence  of  the  very  large 
pyramidal  cells.  In  the  gyrus  hippocampi  or  uncinate  gyrus  there 
are  other  peculiarities,  its  grey  matter  consisting  of  numerous 
conical  cells  with  very  long  processes.  It  is  hardened  in  the  same 


CEREBRUM. 


355 


XXXI.] 

way.  The  peculiar  shape  of  the  cells  in  this  region  is  best  shown 
by  the  “double-impregnation”  method  of  Kamon  y Cayal  (p.  222). 

7.  Blood-Vessels  of  the  Cerebrum  (L  and  H). — Make  rather 
thick  sections  of  an  injected  brain,  and  mount  them  in  balsam. 
They  are  best  embedded  and  cut  in  paraffin.  The  whole  head 
should  be  injected  from  the  aorta. 

(a.)  (L)  The  larger  vessels  in  the  pia  mater  send  into  the  cerebral 
cortex  two  sets  of  arteries : those  that  perforate  tlie  grey  matter 
and  proceed  to  supply  the  medulla  — the  long  or  medullary  arteries  ; 
and  a more  numerous,  shorter  set,  that  ramify  chiefly  iji  the  grey 
matter — the  short  or  cortical  arteries.  The  grey  matter  is  much 
more  vascular  than  the  white,  and  the  vessels  are  surrounded  by 
perivascular  sheaths  (fig.  329). 

(h.)  Study  the  arrangement  and  relative  vascularity  of  the  capil- 
lary plexus  in  the  cortex.  At  the  surface  it  is  less  dense,  and  it  is 
most  dense  in  the  region  of  the  large  pyramidal  cells. 

The  best  resume  of  the  researches  of  Golgi,  R.  y Cayal,  and 
Kolliker  is  given  by  Waldeyer,i  whose  papers  contain  numerous 
woodcuts  showing  not  only  the  histological  results,  but  their  bearing 
on  physiological  problems. 


ADDITIONAL  EXERCISES. 

8.  Cerebellum  in  Osmic  Acid. — Place  very  small  pieces  of  the  grey  matter 
(i  mm.  cubes)  in  i per  cent,  osmic  acid  (24-48  hours)  as  recommended  for  the 
cerebrum  (Lesson  XXXI.  13).  Wash  in  water  and  harden  in  alcoliol.  In  the 
sections  note  the  medullated  fibres  in  the  granular  layer,  but  none  of  them 
pass  into  the  outer  layer  of  the  cortex. 

9.  Cerebellum  in  Ehrlich-Biondi’s  Fluid. — Stain  sections  in  this  fluid  as 
directed  under  Spinal  Cord  (Lesson  XXX.  13)  and  mount  in  balsam.  The 
outer  grey  layer  and  the  medulla  are  red,  the  granular  layer  violet.  In  the 
latter  can  be  seen  the  two  kinds  of  cells,  one  stained  red,  the  other  violet. 

10.  Purkinje’s  Cells  by  Golgi’s  Method  (p.  344). — {a.)  The  easiest  method 
is  the  slow  silver  nitrate  one  (p.  344).  If  the  preparation  is  successful,  one  is 
rewarded  by  the  beauty  of  the  preparation.  It  is  almost  impossible  to 
reproduce  the  complexity  of  the  processes  of  Purkinje’s  cells  (fig.  330).  They 
are  stained  black,  the  nerve-fibres  may  or  may  not  be  stained.  The  sections 
are  mounted  in  balsam  without  a cover-glass.  The  much-branched  proto- 
plasmic processes,  or  dendrites^  are  shown  in  fig.  330,  A,  as  they  appear  when 
a section  is  made  across  the  laminae,  and  in  B when  the  section  is  in  the 
direction  of  the  laminae.  The  dendrites  do  not  anastomose  with  each  other, 
and  all  lie  in  one  plane  in  the  transverse  direction  of  the  leaflet. 

ijb.)  Fibres  in  Cerebellar  Grey  Matter. — By  using  the  rapid  hardening 
method  of  Golgi  and  Cayal  many  other  details  may  be  made  out,  but  not 
all  necessarily  in  one  section.  Round  the  body  of  each  cell  of  Purkinje  is 
a plexus  of  fibrils — basket-work  of  fibres — produced  by  the  division  or  arbori- 

^ Deutsche  med.  fVochensch.,  Xos  44,  45,  46,  47,  1891. 


356 


PRACTICAL  HISTOLOGY. 


[XXXI. 


sation  of  the  axis-cylinder  processes  of  nerve-cells  lying  in  the  molecular 

layer  of  the  grey  matter. 
{'illiliMlt!  'iVjilvtrW  The  dendrites  are  in  part 
also  invested  by  a basket- 
work  due  to  the  arborisa- 
tion of  fibres  proceeding 
from  the  medulla — f.c,, 
fibres  which  do  not  ter- 
minate in  the  cells  of 
Purkinje.  Some  of  the 
cells  of  the  granular 
layer  are  nerve-ctdls  with 
protoplasmic  and  axis- 
cylinder  processi's:  The 

) T small  nerve-cells,  by  far 

j the  most  numerous,  give 

axis  - cylinder  processes 
wliicb  run  out  into  the 
molecular  layer,  where 
they  divide  and  become 
continuous  with  a fibre 
running  at  right  angles 
to  it.  In  the  molecular 
layer  there  is  an  arrange- 
ment of  nerve-fibres  run- 
A.  Seen  ning  more  or  less  parallel 
to  the  surface  of  the 
leaflets,  and  joined  here 

and  there  by  the  axis-cylinder  processes  of  the  nerve-cells  of  the  granular 


Fig.  330.-- -Cell  of  Purkinje.  Corrosive  sublimate. 

on  the  flat,  and  B,  from  the  side,  x 120. 


Fig.  331. — Scheme  of  T.S.  of  Cerebrum  of  New-born  Rat,  prepared  by  Cayal’s  Double- 
impregnation Method.  A.  Corpus  callosum  ; c.  Lateral  ventricle  ; R.  ant^ro-posterior, 
or  association  fibres  arising  from  large  pyramidal  cells ; a.  Large  pyramidal  cells  whose 
axis-cylinder  processes  pass  into  the  antero-posterior  layer ; h.  Fibre  of  corpus  cal- 
losum bifurcating  ; c.  Callosal  fibre  ; d.  Callosal  fibre  arising  from  a pyramidal  cell ; 
e.  Axis-cylinder  process  descending  obliquely  to  enter  the  corpus  callosum ; /.  Final 
ramification  of  a callosal  fibre  in  the  grey  matter  of  the  cortex ; h.  Collateral  fibre 
from  a large  pyramidal  cell ; i.  Fusiform  cells  with  axis-cylinder  processes  passing  into 
outer  layer  of  cortex  ; j.  Final  ramification  of  a callosal  fibre  arising  in  opposite  side 
of  cortex. 


XXXI.] 


CEREBRUM. 


357 


layer  already  described.  If  the  cerebellar  lamina  be  cut  in  a direction  trans- 
verse to  the  course  of  these  fibres,  then  they  merely  appear  as  black  dots. 
My  experience  leads  me  to  believe  that  this  system  is  best  demonstrated  by 
the  rapid  Golgi  method. 

11.  Cerebrum  by  Golgi’s  Method  (see  Lesson  XXX.  19). — Do  the  same  as 
for  the  cord  and  cerebellum.  Either  the  AgXOg  or  HgClg  methods  may  be 
adopted.  In  the  latter  case  the  best  results  are  obtainecl  by  keeping  the  por- 
tions of  the  brain  for  months  in  the  mercuric-chloride 
solution.  I have  usually  got  good  results  with  the 
brain  of  the  rat  or  rabbit.  In  all  cases  mount  the  sec- 
tions in  balsam  without  a cover-glass.  In  new-born 
animals  better  results  are  obtained — especially  with  the 
rapid  method  (osmico-bichromate  method,  p.  345) — than 
with  adult  brains.  Fig.  331  shows  some  of  the  results 
which  may  be  obtained  in  a T.  S.  of  the  brain  of  a new- 
born rat.  The  results  have  been  pieced  together  from 
a study  of  many  sections. 

By  the  rapid  Golgi  method  it  is  easy  to  obtain  beauti- 
ful preparations  showing  the  pyramidal  cells  of  the 
cortex  with  their  apical,  lateral,  and  axis-cylinder  pro- 
cesses ; in  fact,  I have  often  obtained  such  in  the 
superficial  sections  after  a few  hours’  immersion  in 
silver  n itrate.  For  the  cornu  ammonis  cells  the  double- 
impregnation method  of  K.  y Cayal  is  excellent.  Some- 
times. however,  one  gets  the  blood-vessels  stained. 

12.  Cerebrum  by  Weigert  or  Weigert-Pal’s  Method 
(see  Lesson  XXX.  4). — This  method  reveals  the  exist- 
ence of  medullated  fibres  in  the  cerebral  cortex  arranged 
according  to  the  scheme  shown  in  fig.  332. 

At  the  lower  pait  of  the  grey  matter  a large  number 
of  fine  medullated  nerve-fibres  enter  it — radii — which 
run  outwards  in  the  cortex  as  medullary  rays.  Between 
the  latter  is  an  inter-radial  plexus,  and  above  them  is 
a supra-radial  plexus,  and  quite  under  thepia  are  tan- 
gential fibres.  On  the  boundary-line  between  the  supra- 
radial  plexus  and  the  inter-radial  plexus  is  a white 
stripe,  visible  to  the  naked  eye,  especially  well  marked 
in  the  cuneus.  It  has  been  called  the  stripe  of  Gennari, 

Baillaiger,  and  also  of  Vicq  d'Azyr. 

13.  Medullated  Nerve-Fibres  in  the  Cortex  Cerebri. 

— Place  verysniall  pieces  of  the  outer  part  of  the  cortex 
in  I per  cent,  osmic  acid  (24  hours).  The  pieces  must 
be  black  throughout,  ^lake  thin  sections,  place  one 
on  a slide,  add  a drop  of  ammonia  ; the  sestion  swells 
up  and  the  medullated  fibres  become  distinct.  Expose 
the  section  to  the  vapour  of  osmic  acid,  i.e.,  place  the 
section  on  a slide  over  a glass  thimble  filled  with  osmic 
acid  and  cover  the  whole  with  a bell-jar.  After  half  an  hour  or  less  the 
“fixation”  is  complete.  This  is  Exner’s  method  as  modified  by  Ranvier. 
Preserve  in  glycerine.  Observe  the  narrow  medullated  nerve-fibres  between 
the  nerve-cells  ; the  nuclei  only  of  the  latter  are  distinctly  seen. 

Hypophysis  Cerebri  or  Pituitary  Body. 

14.  Hypophysis  Cerebri. — This  consists  of  two  parts,  the  one  derived  from 
the  brain,  a continuation  of  the  infundibulum  ; the  lower  part  is  derived  from 


Fig.  332.— V.S.  Cortex 
Cerebri.  The  right 
side  drawn  fiom  a 
Weigert’s  haema- 
toxylin  preparation, 
and  the  left  from  a 
Golgi’s  sublimate 
one.  On  the  right 
the  medullated 
fibres,  and  on  the 
left  only  the  nerve- 
cells  are  shown. 
There  are  really 
more  cells  than 
shown  in  the  draw- 
ing. 


358 


PRACTICAL  HISTOLOGY. 


[XXXII. 


an  inflexion  of  the  embryonic  mucous  membrane  of  the  mouth.  Harden  in 
Muller’s  fluid,  stain  sections  in  picro-carmine,  and  mount  in  balsam.  Observe 
sections  of  closed  gland-tubes  or  alveoli  almost  completely  filled  with  cubical, 
somewhat  granular,  or  clear  cells.  A lumen  is  rarely  visible.  The  communica- 
tion between  the  mouth  and  the  lower  part  of  the  gland  is  cut  off  in  the  pro- 
cess of  development. 


LESSON  XXXII. 

THE  EYE. 

Methods. — (i.)  Enucleate  the  eyeball  of  a rabbit  or  cat;  remove 
any  adhering  fat  and  muscles.  With  a sharp  razor  make  a single 
cut  at  the  equator  of  the  eyeball,  and  suspend  it  in  200  cc.  of  .25 
per  cent,  chromic  acid.  After  twenty-four  hours  cut  the  eye  into 
an  anterior  and  a posterior  half,  and  place  them  for  several  days  in 
new  fluid.  Wash  and  harden  in  the  dark  in  gradually  increasing 
strengths  of  alcohol.  The  eye  hardened  in  this  way  may  be  used 
for  preparing  sections  of  some  of  its  parts. 

(ii.)  Harden  the  other  eyeball  for  two  or  three  weeks  in  Miiller^s 
fluid,  after  cutting  into  it  in  the  same  way  as  above.  Complete  the 
hardening  in  alcohol.  It  is  well  to  have  sections  of  the  cornea  of 
several  animals,  e.g.^  the  pig  and  ox. 

THE  CORNEA. 

Make  Y.S.  from  a cornea  hardened  in  MiilleEs  fluid  or  Flemming’s 
fluid.  Stain  with  picro-carmine  and  mount  in  Tarrant’s  solution, 
or  in  hsematoxylin  and  mount  in  balsam.  Stain  the  one  fixed  in 
Flemming’s  fluid  with  safranin.  A good  method  is  to  stain  it  in 
bulk  in  borax-carmine.  Eosin-haematoxylin  is  a good  stain. 

1.  V.S.  Cornea  (L  H.). 

(a.)  Observe  the  anterior  or  conjunctival  epithelium,  consisting 
of  several  layers  of  stratified  epithelium.  The  most  external  cells 
are  flattened  ; those  of  the  middle  layers  are  more  oval  or  rounded, 
many  of  them  with  finger-shaped  processes  dipping  down  between 
the  deeper  rows  of  cells.  The  cells  in  the  lowest  layer  are  columnar, 
and  placed  perpendicularly  upon  the  cornea,  resting  on  (h) 
(fig-  333)- 

{h.)  The  narrow,  clear,  transparent  layer — anterior  elastic 
lamina ; this  is  best  marked  in  the  human  eyeball. 

(c.)  The  substantia  propria,  or  body  of  the  cornea,  composed  of 


XXXII.  ] 


THE  EYE. 


359 


layers  of  transparent  fibrous  tissue  arranged  in  laminae.  The 
laminae,  while  arranged  in  the  main  parallel  to  each  other,  have 
connecting  processes  joining  adjacent  laminae.  Between  the  laminae 
oval  spaces,  and  in  each  space  a nucleated  cornea  corpuscle  (fig. 
333,  c),  which,  as  seen  on  edge,  is  thin  and  flattened.  The  processes 
of  these  corpuscles  are  best  seen  in  gold  specimens. 

{d. ) The  posterior  elastic  lamina,  or  membrane  of  Descemet, 
a well-marked,  thin,  transparent,  hyaline  lamina,  with  sharply 
defined  outlines,  and  covered  posteriorly  by 

{e.)  The  posterior  epithelium,  consisting  of  a single  layer  of 
large  flattened  nucleated  cells,  seen  in  profile.  They  are  apt  to  be 
displaced  if  the  section  be  roughly  handled. 

If  the  posterior  elastic  lamina  be  broken  across,  it  is  apt  to  curl 
up.  It  stains  readily  with  carmine. 


Fig.  333.— V.S.  Cornea,  a.  Epi- 
thelium ; h.  Anterior  elastic 
lamina;  c.  Corneal  corpuscles; 
1.  Lamellae  of  cornea ; 
Substantia  propria ; d.  I)es- 
cemet’s membrane;  and  e.  Its 
epithelium. 


2.  Herves  of  the  Frog’s  Cornea. — In  a pithed  frog,  squeeze  the 
head  to  make  the  eyeball  jiroject,  and  with  a sharp  razor  cut  off  the 
cornea.  Wash  it  in  normal  saline,  treat  it  with  gold  chloride  by 
the  lemon-juice  method  (p.  79). 

After  impregnation  with  the  gold  solution,  with  scissors  make 
three  snips  into  its  margin,  dividing  it  into  three  sections,  so  that 
it  may  lie  flat  on  a slide.  Mount  in  Tarrant’s  solution. 

(«.)  (L  and  H)  Observe  the  branching  cornea  corpuscles, 
arranged  in  many  layers,  one  under  the  other,  readily  seen  by 
raising  or  depressing  the  lens  (fig.  334).  The  numerous  processes 
given  off  from  the  cells  anastomose  with  similar  processes  from 


PRACTICAL  HISTOLOGY. 


360 


[XXXII. 


adjoining  cells,  not  only  in  the  same  plane,  but  with  cells  lying  in 
planes  above  and  below  them. 

(6.)  The  Nerves. — If  a part  of  the  sclerotic  is  adherent  to  the 
cornea,  the  fine  medullated  nerve-fibres,  deeply  stained,  are  seen  in 

the  sclerotic  and  passing 
into  the  cornea,  where 
they  lose  their  myelin 
and  become  non-medul- 
lated.  The  larger  non- 
medullated  fibres  have 
nuclei  in  them,  and  unite 
with  other  fibres  to  form 
a coarse  plexus  — the 
ground  or  primary  nerve 
plexus, 

(c.)  From  this  plexus 
finer  bundles  of  fibrils 
proceed  to  form  a finer 
plexus,  and  from  the 
latter  numerous  fibrils — 
often  with  varicose  swel- 
lings— running  chiefly  in  the  planes  of  the  laminae  are  seen. 

{d.)  (H)  Fine  fibrils  are  seen  in  the  larger  branches  of  the  non- 
medullated  nerves  (fig.  335). 

If  the  cornea  of  a rabbit  be  used,  then  tangential  thin  sections 

must  be  made  and  mounted 

J ^ 


Fig  335.— Sub-Epithelial  Nerve-Flexus,  Cornea  of 
Frog.  n.  Non-inedullated  nerve  with  nerve- 
fibrils  ; a.  Nerve-fibrils,  x 300. 


in  Farrant^s  solution  or 
balsam. 

3.  Inter-Epithelial  Ter- 
mination of  the  Nerve- 
Fibres. — The  cornea  of  a 
rabbit  is  stained  in  i per 
cent,  gold  chloride  (25-30 
minutes)  and  reduced  in 
slightly  - acidulated  water 
(acetic  acid)  by  exposure  to 
light  (p.  79).  Tlie  lemon- 

FiG.  336.— V.S.  Cornea  of  Frog.,  n.  Nerve-fibres;  juice  and  formic-acid  method 
a.  Perforating  fibrils;  r.  Nucleus ; Inter-  , ■ j.  u a 

epithelial  plexus  of  fibrils.  Gold  chloride.  muSt  not  be  USed,  aS  tue 

formic  acid  removes  the 
epithelium.  Make  Y.S.  either  by  freezing  or  free  hand  and  mount 
them  in  glycerine  (L  and  H). 

{a.)  Observe  sections  of  the  lamellae  and  corneal  corpuscles,  and 
between  these,  parts  of  the  primary  nerve-plexus  cut  across,  more 
or  less  obliquely,  the  branches  being  finer  towards  the  anterior 


XXXII.] 


THE  EYE. 


361 


elastic  lamina.  Bundles  of  fine  fibrils— the  rami  perforantes — 
perforate  this  membrane  and  spread  out  under  the  anterior  epi- 
thelium to  form  the  sub-epithelial 
plexus,  from  which  fibrils  proceed  to 
form  a plexus  of  fine  varicose  fibrils — 
the  epithelial  plexus  — between  the 
epithelial  cells,  where  they  terminate 
in  fine  points,  forming  no  connection 
with  special  end-organs  (fig.  336). 

4.  Cell-Spaces  in  the  Cornea  (Silver 
Method)  ( L and  H).  — Pith  a frog,  scrape 
off  the  corneal  epithelium,  and  rub  the 
surface  of  the  cornea  with  solid  silver 
nitrate.  After  20-30  minutes,  excise 
the  cornea,  mount  it  in  glycerine,  and 
expose  to  light. 

(a.)  The  general  substance  of  the 
section  is  stained  brown,  and  in  it  are 
clear  branching  spaces,  each  one  corresponding  in  shape  to  that  of 


Fig.  337  —Cornea  of  Frog  with  Cell- 
Spaces.  s Cell-spaces.  Solid 
silver  nitrate,  x 300. 


Fig.  338. — V.S.  Part  of  Sclerotic  and  the  Choroid,  g.  Large  vessels;  p.  Pigment-cells; 
c.  T.S.  of  capillaries,  x 100. 


a cornea  corpuscle  (fig.  337).  The  branches  of  adjoining  spaces 
anastomose,  so  as  to  form  a system  of  juice-canals,  not  only  with 
32 


362 


PRACTICAL  HISTOLOGY. 


[XXXII. 

spaces  in  the  same  plane,  but  also  with  the  spaces  in  lower  and 
deeper  planes. 

5.  Iron  Sulphate  Method  (Lesson  XI.  13).— The  matrix  is  blue 
and  the  spaces  are  clear  and  not  stained. 


THE  SCLEKOTIC,  CHOEOID,  AND  CILIARY  REGION. 

6.  Sclerotic  and  Choroid  (L,.H.).  -From  an  eye  hardened  in 
Muller  s fluid  and  stained  in  hulk  in  horax-carmine  cut  sections  in 

paraffin  to  include  the  sclerotic  and  choroid.  Observe 

(i.)  The  sclerotic  (fig.  338).  {a.)  Composed  mainly  of  bundles 

of  white  fibrous  tissue  crossing  each  other  in  several  directions. 


Fig.  339.— Horizontal  Section  of  Anterior  Quadrant  of  Eyeball.  /.  Conjunctiva;  s. 
Sclerotic  ; h.  Iris;  j.  Lig.  pectinatum  iridis ; k.  Canal  of  Schlernm ; 1.  Longitudinal; 
m.  Circular  fibres  of  ciliary  muscle ; n.  Ciliary  process ; o.  Ciliary  part  of  retina. 


(h,)  A narrow  layer,  the  lamina  supra-choroidea. 

(2.)  Inside  this  a section  of  the  choroid  composed  of  the  follow- 
ing layers  from  without  inwards — 

(a.)  Layer  with  large  vessels  and  numerous  branched  pigment- 
cells.  This  forms  by  far  the  thickest  layer. 

(h,)  Limiting  layer. 


xxxil]  the  eye.  363 

(c.)  The  chorio-capillaris,  containing  numerous  sections  of  capil- 
laries. 

{d.)  A basement  or  hyaline  membrane. 

(e.)  Pigment-cells  containing  melanin. 

7.  Pigment-Cells  of  the  Choroid  (L.H.). — From  the  inner  cho- 
roidal surface  of  an  eye  hardened  in  Muller’s  fluid  or  spirit,  scrape 
olf  a little  of  the  black  pigment  layer  and  mount  it  in  balsam. 

(a.)  Observe  the  branched  pigmented  cells,  filled  with  brown  or 
black  granules  of  melanin.  The  nucleus  contains  no  pigment 
granules.  If  the  granules  be  discharged  from  the  cells  and  float  in 
a watery  fluid,  they  exhibit  Brownian  movement.  The  nucleus 
stains  readily  with  haematoxylin. 

8.  Ciliary  Eegion,  Ciliary  Muscle,  and  Iris.  — Make  a meri- 
dional section  through  the  corneo-scleral  junction  of  a hardened  eye 
(Muller’s  fluid),  e.g.^  of  an  ox,  cat,  or  rabbit,  so  as  to  include  the 
ciliary  muscle  and  the  iris.  Stain  the  section  in  hsematoxylin  or 
eosin-hgematoxylin,  and  mount  in  balsam ; or  it  may  be  stained  in 
picro-carmine  and  mounted  in  Farrant’s  solution.  To  preserve  all 
the  parts  exactly  in  position  cut  it  in  celloidin,  or  stain  in  bulk  and 
cut  in  paraffin.  Fix  the  sections  on  a slide  by  a fixative. 

(a.)  Observe  the  cornea  passing  into  the  sclerotic,  and  near  it  an 
opening,  the  canal  of  Schlemm  (fig.  339). 

(b.)  From  the  angle  of  the  iris  and  cornea,  running  in  a fan- 
shaped expansion,  the  radiating  fibres  of  the  ciliary  muscle,  con- 
sisting of  non-striped  muscle.  The  circular  fibres  cut  transversely 
inside  the  radiating  fibres  are  not  so  well  marked  as  in  the  human  eye. 

(c.)  The  membrane  of  Descemet  splits  up  into  fibres,  some  of 
which  curve  into  the  iris,  others  spread  into  the  ciliary  processes. 
This  region  constitutes  the  ligamentum  pectinatum  iridis. 

(d,)  The  pigmented  folds  or  ciliary  processes.  A layer  of  black 
pigment-cells  continuous  with  those  of  the  retina  continued  over 
these  processes,  and  inside  the  pigment  a clear  unpigmented  layer 
of  columnar  epithelium. 

(e.)  The  iris,  composed  of  five  layers  from  before  backwards. 

(i.)  A layer  of  endothelium  continuous  with  that  covering  the 
posterior  surface  of  the  cornea  (fig.  339,  c). 

(2.)  The  anterior  boundary  layer ^ chiefly  consisting  of  branched 
connective  tissue-cells. 

(3.)  The  vascular  layei\  or  body  of  the  iris,  composed  of  a stroma 
of  connective  tissue  with  numerous  sections  of  blood-vessels  and 
branched  pigment-cells,  and  at  its  deeper  part  transverse  sections  of 
the  circular  smooth  muscular  fibres,  which  constitute  the  circular 
muscle  or  sphincter  2mpill(B  of  this  organ.  It  is  doubtful  if  a 
dilator  pupillae  exists  in  man. 

(4.)  The  posterior  hyaline  layer,  of  an  elastic  nature. 


364 


PRACTICAL  HISTOLOGY. 


[XXXII. 


(5.)  Two  layers  of  pigment-cells — the  uvea — the  outlines  of  the 
cells  difficult  to  define,  and  continuous  with  the  epithelium  of  the 
pars  ciliaris  retinae. 

(6.)  Blood-Vessels. — A large  number  of  blood-vessels  and  nerves 
pass  into  the  iris,  but  their  distribution  must  be  studied  by  the 
methods  already  described  for  such  purposes. 

THE  LENS. 

The  lens  is  developed  by  the  inflection  of  part  of  the  epiblast,  so 
that  it  consists  of  modified  epiblastic  epithelial  cells  invested  by  a 
transparent  capsule.  The  capsule  of  the  lens  is  a clear  trans- 
parent elastic  membrane,  not  unlike  a basement  membrane,  but  is 
less  resistant  to  acids  than  the  latter.  Immediately  on  the  inner 
surface  of  its  anterior  part  is  a layer  of  clear  flattened  epithelial 
cells — hexagonal  and  nucleated — the  remains  of  the  original  cells. 
Tracing  these  cells  towards  the  margin  of  the  lens  they  become 
narrower  and  more  cylindrical,  until  further  on  they  gradually  pass 
into  lens  fibres.  Posteriorly  there  are  no  epithelial  cells  between 
the  lens  and  its  capsule,  the  lens  fibres  resting  directly  on  the 
capsule  itself.  The  lens  fibres  are  modified  epithelial  cells,  the 
fibre  constituting  the  principal  part  of  the  cell.  The  cells  are  long, 
soft,  hexagonal  prisms  united  to  each  other  by  more  or  less  toothed 
edges.  The  superficial  iibres  are  longer  and  larger  than  the  deeper 
ones,  and  contain,  or  have  on  their  surface,  nuclei  lying  on  the  part 
that  corresponds  to  the  equatorial  (nuclear)  zone  of  the  fibre.  No 
nucleus  is  visible  in  the  central  fibres. 

9.  Methods. — Harden  in  its  capsule,  the  lens  of  a cat  or  rabbit, 
in  Muller’s  fluid  (2-3  weeks) ; do  not  place  it  in  spirit,  but  cut  it 

in  a freezing  microtome,  or  cut  in  cel- 
loidin.  Paraffin  is  not  suitable.  Stain 
the  sections  in  eosin  and  mount  in 
Farrant’s  solution.  Make  meridional 
and  equatorial  sections. 

T.S.  Lens — (a.)  (L.H.).  — Observe 
the  hyaline  capsule — if  present — and 
just  under  its  front  part  a layer  of 
epithelial  cells,  which,  when  traced 
outwards,  are  seen  to  rapidly  elongate 
into  lens-fibres  (p.  364).  Nuclei  may 
be  seen  on  some  of  the  fibres. 

(b.)  T.S.  of  the  fibres  are  hexagonal 
(fig.  340,  B). 

10.  Isolated  Lens  Fibres — Methods. — (a.)  Place  the  lens  of  a 
frog  or  fish  in  a 10  per  cent,  solution  of  sulphocyanide  of  potas- 


THE  EYE. 


365 


XXXII.] 

sium  (24-36  hours).  Tease  out  a fragment  of  the  softened  lens  in 
glycerine,  (h.)  Or  use  dilute  sulphuric  acid  (4-5  drops  to  5 cc.  of 
water  for  24-36  hours).  Wash  in  water  to  remove  the  acid.  Stain 
in  carmine  or  haematoxylin.  (c.)  Methyl-green  feebly  acid,  does  well 
for  fresh-teased  fibres,  but  the  preparations  do  not  keep.  The 
nuclei  on  the  fibres  may  be  seen  in  this  preparation. 

(H)  (a.)  Observe  the  flattened  bands  with  serrated  edges ; the 
teeth  dove-tail  into  each  other.  The  teeth  may  be  seen  from  the 
side  or  directed  to  the  observer. 

EETINA. 

Methods. — (i.)  With  a slkarp  razor  cut  the  eye  of  a cat,  rabbit, 
pig,  or  other  animal  into  an  anterior  and  posterior  half.  Place  the 
posterior  half  in  2 per  cent,  potassic  bichromate,  or  in  a mixture  of 
Muller’s  fluid  and  spirit  (two  weeks),  and  complete  the  hardening 
in  spirit. 

(ii.)  Stain  ‘On  bulk”  in  borax-carmine  for  several  days  the 
whole  posterior  segment  of  the  hardened  eyeball,  then  place  it  for 
a day  in  acid  alcohol,  and  embed  in  paraffin  and  cut  sections.  Per- 
haps celloidin  is  to  be  preferred  if  the  sclerotic  be  thick.  Fix  the 
sections  on  a slide  and  mount  in  balsam.  In  this  way  the  whole 
structure  of  the  retina  is  preserved  in  its  relations  to  other  struc- 
tures. 

(iii.)  The  same  process  may  be  adopted  with  the  posterior  seg- 
ment of  the  eyeball  hardened  in  i per  cent,  osmic  acid. 

(iv.)  Harden  the  retina  in  a 3 per  cent,  solution  of  nitric  acid 
(15-20  mins.).  Wash  out  the  acid,  stain  in  bulk,  and  make  sections. 
Stain  with  eosin  and  methylene-blue. 

(v.)  For  small  eyes,  such  as  those  of  a lizard,  it  is  sufficient  to 
expose  the  whole  unopened  eyeball  to  the  vapour  of  osmic  acid  for 
several  hours,  and  then  to  stain  the  eyeball  in  bulk  in  borax-carmine. 
Embed  in  paraffin  and  cut  sections  of  the  whole  eyeball.  Or,  expose 
the  eye  of  a frog  or  triton  to  osmic  acid  vapour  (10  mins.).  Place 
in  40  per  cent,  alcohol  (4-6  hours) ; divide  eyeball  by  incision 
parallel  to  edge  of  cornea ; bichromate  of  ammonia  (3  per  cent.)  for 
5-10  hours;  wash  in  water;  alcohol;  borax-carmine;  embed  and 
cut  in  paraffin. 

(vi.)  Make  sections  of  an  eyeball  hardened  in  Muller’s  fluid  and 
stain  them  in  eosin-haematoxylin.  The  latter  stains  the  granular 
layers  of  the  retina,  while  the  other  parts  are  rosy  in  tint. 

(vii.)  Methylene-Blue  Method. — This  is  done  either  by  injecting 
methylene-blue  (i  in  300  normal  saline)  into  the  blood-vessels  of  an 
animal  just  killed,  and  waiting  two  or  three  hours  thereafter,  or  by 
placing  the  fresh  retina  in  the  same  fluid  and  observing  with  the 


366 


PRACTICAL  HISTOLOGY. 


[XXXII 


microscope  the  coloration  of  the  nervous  elements,  especially  the 
cells  in  the  retina.  Mount  in  picrate  of  ammonia  and  glycerine  (p. 
192). 

11.  V.S.  Retina. — (L)  Beginning  from  within,  ^.e.,  next  the 
vitreous,  observe  (fig.  341) — 

{a, ) The  internal  limiting  membrane, 
and  springing  from  it  by  wing-shaped 
expansions  the  fibres  of  Muller,  which 
run  vertically  outwards  through  the 
layers  of  the  retina  to  the  outer  limiting 
membrane. 

{b.)  The  fibrous  layer,  composed  of 
the  non-mediillated  fibres  of  the  optic 
nerve.  The  fibres  are  medullated  in  the 
optic  nerve,  but  they  lose  their  medulla 
as  they  enter  the  retina.  The  layer  is 
thinner  in  the  anterior  part  of  the 
retina.  Some  of  the  nerve-fibres  end 
in  the  ganglionic  cells  of  the  next  layer, 
but  others  pass  outwards  to  the  inner 
molecular  layer  to  end  by  terminal 
arborisations  there  (shown  by  Golgi’s 
method). 

(c.)  The  ganglionic  or  nerve-cell 
layer,  consisting  of  a single  row  of  large 
multipolar  nerve-cells,  each  cell  with  a 
large  conspicuous  nucleus.  Each  cell 
gives  off  (i.)  one  thick  process,  which 
may  divide  into  several  processes  which 
pass  towards  and  ramify  by  terminal 
arborisations  in  the  layer  external  to  this, 
and  (ii.)  one  fine  axis-cylinder  process 
the  optic  nerve -fibre  layer, 
where  it  becomes  continuous  with  a 
nerve-fibre.  Between  the  nerve-cells,  or 
in  close  relation  to  them,  sections  of  the 
larger  blood-vessels. 

{d.)  The  internal  molecular  layer 
appears  to  consist  of  fine  fibrils  or 
granules.  It  is  not  unlike  the  grey  matrix  of  the  cerebrum,  and  is 
traversed  by  the  fibres  of  Muller  and  also  by  fibres  of  the  optic  nerve, 
and  processes  of  the  cells  of  the  ganglionic  and  inner  granular  layers^ 
{e.)  The  internal  nuclear  layer,  deeply  stained,  and  consisting 
of  several  rows  of  large  spherical  oval  nuclei. 

Some  of  the  granules,  however,  l)y  other  methods  can  be  shown 


RC 


EN 


EG 


IN 


IG 


Fig.  341. — V.S.  Retina  of  Cat.  h. 

Rods  and  cones ; le,  li.  Exter- 
nal and  internal  limiting  mem- 
branes ; nb.  Nuclei  of  rods  ; 
pb.  Basal  plexus ; cb.  Bipolar 
cells  ; cu.  Unipolar  cells  ; bi. 

Internal  basal  cells  ; pc.  Cere- 
bral plexus;  cm.  Multipolar 
nerve-cells ; fo.  Fibres  of  optic  Wdias 
nerve.  On  the  left  the  initials 
of  the  usual  names  for  these 
layers ; RC.  Rods  and  cones  ; 

EN.,  EG.,  and  IN,  IG.  Ex- 
ternal and  internal  nuclear 
and  granular  layers;  C.  Cellular 
layer ; F.  Fibrous  layer. 


XXXII.] 


THE  EYE. 


367 


to  be  bipolar  cells  with  large  nuclei.  The  processes  extend  into  the 
inner  molecular  layer  and  end  in  arborisation,  and  by  Golgi’s  method 
the  opposite  processes  have  been  traced  outwards  as  far  as  the 
external  limiting  layer.  Other  branched  cells  supposed  to  resemble 
neuroglia  cells  lie  amongst  these  cells. 

(/.)  The  outer  molecular  layer  is  thin,  and  is  composed  of  the 
terminal  arborisations  of  processes  from  the  rods  and  cones  and 
inner  granular  layer. 

(y.)  The  external  nuclear  layer  consists  of  many  more  rows  of 
nuclei  than  in  (c).  They  are  the  nuclei  of  the  rod  and  cone  fibres. 

(h.)  The  external  limiting  membrane,  cut  across,  and  therefore 
appearing  as  a thin  clean-cut  line. 

(/.)  The  layer  of  rods  and  cones.  The  rods  are  more  numerous 
than  the  cones,  and  the  latter  are  shorter  than  the  former.  Each 
rod  and  cone  consists  of  an  outer  and  an  inner  segment. 

(/)  A layer  of  hexagonal  pigment-cells,  somewhat  flattened, 
which  sends  pigmented  processes  or  filaments  between  the  rods  and 
cones.  The  length  and  condition  of  these  processes  depends  upon 
the  influences  to  which  the  eye  has  been  exposed  before  death 
(p.  370).  The  pigment  granules,  some  of  which  are  crystalline, 
exist  chiefly  in  the  inner  part  of  the  cells,  and  may  extend  into  the 
cell  processes  which  pass  between  the  rods  and  cones. 

(H)  Study  the  successive  layers.  Although  the  layer  of  rods 
and  cones  and  external  nuclear  layer  have  been  described  as 
separate  layers,  in  reality  they  should  be  studied  together.  Each 
rod  is  continued  inwards  by  a tapering  fibre — the  rod-fibre  ; in  the 
course  of  the  latter  is  an  oval  nucleus,  the  fibre  itself  ending  in  an 
arborisation  in  the  outer  molecular  layer.  Each  cone  is  similarly 
prolonged  into  a cone-fibre — thicker  than  the  rod-fibre — which  also 
has  an  enlargement  in  it  containing  a nucleus.  The  cone-fibre  ends 
like  the  rod-fibre  in  the  outer  molecular  layer.  These  two  kinds  of 
nuclei  intercalated  in  the  course  of  the  rod  and  cone  fibres  make  up 
the  external  nuclear  layer,  which  as  the  rods  and  cones  are  merely 
modified  epithelial  cells — must  be  regarded  as  epithelial  in  their 
origin.  In  a freshly -teased  retina  the  nuclei  of  the  rod-fibres  are 
marked  by  alternate  transverse  light  and  dim  stripes. 

Each  rod  and  cone  consists  of  two  segments.  The  outer  segment 
of  the  rods  is  clear  hyaline  and  transparent,  and  striated  trans- 
versely, and  readily  breaks  up  into  transverse  discs.  During  life 
the  external  segments  of  the  rods  contain  the  visual  purple.  The 
inner  segment  is  wider,  granular  and  striated  longitudinally.  The 
cones  also  consist  of  a shorter  tapering  outer  segment  transversely 
striated,  and  an  inner  bulging  larger,  longitudinally  striated,  segment 
which  is  continued  through  the  external  limiting  membrane  into 
the  cone-fibre. 


368  PRACTICAL  HISTOLOGY.  [XXXIL 

The  outer  segments  are  stained  black  by  osmic  acid,  and  the 
inner  segments  red  by  carmine. 

Study  successively  the  other  layers  to  make  out  some  of  the 
details  already  described  on  p.  366. 

12.  Eetina  of  Frog. — (i.)  Place  the  posterior  segment  of  the 
eyeball,  with  its  vitreous  humour  removed,  in  i per  cent,  osmic 
acid  (24  hours);  wash  well,  and  tease  a fragment  of  the  now 
blackened  retina  in  glycerine.  Observe 

{a)  The  rods,  each  with  an  outer  and  inner  segment;  the  outer 
segment,  however,  is  blackened  by  the  osmic  acid,  and  usually 
shows  a tendency  to  split  transversely. 

(/;.)  In  the  cones  there  is  a small  refractive  oil  globule  between 
the  outer  and  inner  segments.  It  first  becomes  brown,  and  then 
black,  in  osmic  acid.  In  some  animals,  e,g.^  some  birds  and 
reptiles,  the  oil  globule  is  pigmented,  the  pigment  being  held  in 
solution  by  the  fat  of  the  globule. 

(c.)  Numerous  pigment  cells,  each  consisting  of  a rather  thick 
body,  part  of  which  contains  the  nucleus  and  is  non-pigmented, 
while  the  other  half  is  pigmented,  and  sends  numerous  fine  pro- 
cesses between  the  outer  segments  of  the  rods. 

(d.)  Other  parts  of  the  retina  are  also  isolated,  e.g.,  the  nuclei  of 
the  nuclear  layer  and  the  limiting  membranes. 

(ii.)  Fix  the  whole  eyeball  in  osmic  acid,  and  make  V.S.  of  all 
the  coats  after  embedding  in  paraffin  or  celloidin. 

13.  Cones. — To  see  large  cones,  place  the  fresh  eye  of  a cod  in  2 
per  cent,  potassic  bichromate  (2-3  days)  or  osmic  acid  (24  hours), 
or  isolate  them  by  dilute  alcohol. 

14.  T.S.  Optic  Nerve  (Land  H).  — Place  the  optic  nerve  in  2 per 
ceut.  ammonium  bichromate  (2-3  weeks)  or  in  Flemming’s  mixture 
(10-20  hours).  Make  T.S.  Stain  in  haematoxylin,  picro-carmine, 
or  safranin,  or  by  Weigert’s  method.  Mount  in  balsam.  Double- 
stain some  in  picro-carmine  and  haematoxylin. 

(a.)  Observe  the  sheaths,  thick  and  well-marked,  sending  septa 
into  the  nerve,  and  thus  breaking  it  up  into  small  bundles  of  nerve- 
fibres,  each  surrounded  by  a fibrous  sheath.  In  the  larger  septa 
are  blood-vessels.  The  anastomosing  septa  form  a kind  of  alveolar 
system,  so  that  a section  of  this  nerve  is  readily  distinguished. 

(h.)  The  nerve-fibres  are  all  medullated,  but  they  possess  certain 
peculiarities.  They  are  without  sheath  of  Schwann,  and  the 
fibrous  tissue  runs  parallel  with  the  nerve-fibres. 

(c.)  As  to  the  sheaths,  one  is  continuous  with  the  dura  mater ; 
under  it  is  a space,  the  subdural  space ; inside  is  a prolongation  of 
the  arachnoid  with  subarachnoid  space,  and  a thin  continuation  of 
the  pia  mater. 

If  desired,  make  L.S.  This  enables  one  to  note  the  structure  of 


XXXII.] 


THE  EYE. 


369 


the  nerve-fibres  and  the  arrangement  of  the  connective  tissue.  An 
interstitial  injection  of  osmic  acid  is  a good  method  for  isolating  the 
structural  elements. 

15.  Blood-Vessels  of  Eyeball. — It  is  not  difficult  to  inject  from 
the  aorta  with  a carmine-gelatine  mass  the  blood-vessels  of  the  eye- 
ball of  an  animal.  Select  an  albino  rabbit.  Sections  are  mounted 
in  balsam.  One-half  of  the  injected  eyeball  of  an  albino  rat 
mounted  in  balsam  shows  the  connections,  distribution,  and 
arrangement  of  the  blood-vessels. 

16.  The  eyelids. — Harden  the  eyelids  of  an  infant  in  corrosive 
sublimate  or  .25  per  cent,  chromic  acid  (3  or  4 days)  and  then  in 
alcohol,  or  in  alcohol  alone.  Make  sections,  vertical  to  its  surface 
and  transverse  to  its  long  axis,  which  must  be  rather  thick,  and 
mounted  in  balsam  to  show  the  general  arrangement  of  the  parts. 
Finer  sections  are  stained  in  haematoxylin  or  picro-carmine.  Be- 
sides the  sections  of  hair-follicles,  connective  tissue,  and  orbicularis 
muscle,  note  the  Meibomian  glands. 


ADDITIONAL  EXERCISES. 

17.  Fibrils  of  Cornea. — (a.)  Macerate  the  cornea  in  lime-water,  baryta- 
water,  or  dilute  potassic  permanganate.  (^>.)  A better  method  is  to  dry  the 
cornea,  make  T.S.,  allow  them  to  swell  up  in  water,  and  stain  with  picro- 
carmine. 

18.  Epithelium  of  Lens. — Use  a small  lens  (frog  or  rat),  and  place  it  for  a 
few  minutes  in  AgXOg  (i : 300).  Expose  to  light  in  glycerine,  and  examine. 
Note  on  the  anterior  surface  («.)  silver  lines  bounding  polygonal  areas  cor- 
responding to  the  inter-epithelial  cement.  (A)  Black  granular  converging  lines 
corresponding  to  the  cement  between  the  crystalline  fibres  (best  seen  on 
posterior  surface). 

19.  Other  Methods  for  the  Retina. — By  the  usual  methods  of  hardening  the 
retina,  it  is  impossible  to  make  out  the  connections  between  its  several  elements. 
The  two  methods  which  have  quite  recently  yielded  the  best  results — apart 
from  the  direct  use  of  osmic  acid — are  the  methods  of  Golgi  and  Ehrlich’s 
methylene-blue  method. 

{a.)  Golgi’s  Methods. — Ramon  y Cayal  has  made  a large  number  of  prepara- 
tions both  by  the  silver  method  (p.  344)  and  the  rapid  osmico-bichromate 
method  (p.  345),  especially  on  the  retina  of  birds.  In  this  way  he  has  shown 
that  the  protoplasmic  processes  of  the  nerve-cells  of  the  ganglionic  layer 
ramify  in  the  inner  molecular  layer,  and  end  in  terminal  arborisations  in  several 
planes  in  this  layer.  Several  varieties  of  nerve-cells  with  extensive  terminal 
arborisations  in  the  other  layers  of  the  retina  ai  e also  described  by  him. 

The  following  medium  method  gives  good  results  : — 

(1.)  Place  the  retina  and  sclerotic  in  weak  osmico-bichromate  fluid 
(osmic  acid,  i per  cent.),  2 cc.,  bichromate  of  potash  (3  per 
cent.),  20  cc.,  for  24  hours. 

(2.)  Transfer  to  i per  cent,  silver  nitrate  (24  hours). 

(3.)  A^ain  for  24  hours,  as  in  (i). 

(4.)  Again  for  24  hours  in  silver. 

33  2 a 


i7o 


PRACTICAL  HISTOLOGY. 


[XXXII. 


{h.)  Methylene-Blue  Method.— DogieP  by  means  of  this  method  finds 
that  in  man  llirre  are  more  nervous  elements  present  than  one  has  been  in 
the  habit  of  supposing.  He  arranges  the  nervous  elements  (in  three  layers) 
and  other  layers  as  follows  : — 


Membrana  limitans 
externa. > 

External  reticular 
layer. 

Inner  reticular 
layer. 


Membrana  limitans 
interna. > 


Pigment  epithelium  (I). 
Neuro-epithelial  layer  (II). 
Sub-epithelial  nerve-cells  (a). 
Stellate  nerve-cells  {h). 
Bipolar  nerve-cells  (c). 

Middle  ganglionic  layer. 

Inner  ganglionic  layer. 

Layer  of  nerve-fibres. 


A.  Outer  ganglionio 

layer. 

B.  Layer  of  spongio- 

blasts of  W. 
Muller. 

C. 

D. 


The  neuro-epithelial  layer  embraces  the  layer  of  rods  and  cones  and  subjacent 
external  nuclear  layer.  What  he  calls  the  outer  ganglionic  layer  corresponds 
to  the  inner  nuclear  layer,  and  contains  three  varieties  of  nerve-cells.  The 
middle  ganglionic  layer  corresponds  to  a layer  of  cells — of  which  there  are 
several  varieties  lying  on  the  confines  between  the  internal  granular  (reticular) 
layer  and  the  internal  nuclear  layer.  These  cells  were  called  spongioblasts 
by  W.  Muller,  but  according  to  Dogiel,  they  are  nerve-cells.  The  inner 
ganglionic  layer  corresponds  to  the  cellular  layer. 

20.  Epithelium  of  the  Retina. — Harden  the  eye  of  a frog  in  picvo-sulphuric 
acid  (24  hours),  stain  in  bulk  in  borax -carmine,  and  make  vertical  sections 
after  embedding  in  paraffin. 

21.  Isolated  Elements  of  the  Retina.  — {a.)  Macerate  the  retina  in  Schieffer- 
decker’s  fluid  as  described  for  the  isolation  of  the  cells  of  the  cord  (Lesson  XXX. ), 
using  the  sulphuric  acid  method  to  remove  the  water. 

{h.)  For  Muller’s  fibres  10  per  cent,  chloral  hydrate  is  good. 

22.  Retina  of  Frog  in  Light  and  Darkness.— (a.)  Keep  one  frog  in  abso- 
lute darkness  for  thirty-six  hours.  Kill  it  in  the  dark,  and  harden  the  eye  in 
alcohol,  {h,)  Place  another  frog  in  direct  sunlight  for  a few  hours  ; kill  it, 
and  harden  the  retina  in  alcohol.  Sections  are  made  and  stained  with  picro- 
carmine.  The  pigment-cells  covering  the  rods  of  the  retina  in  {a)  are 
retracted,  while  those  in  {h)  are  pushed  out  between  the  segments  of  the  rods. 

23.  V.S.  Macula  Lutea. — Secure  a human  eye  as  fresh  as  possible.  Harden 

it,  and  make  sections  through  the  macula  lutea,  and  observe  the  peculiarities 
of  its  structure.  There  are  no  rods  in  the  fovea  centralis,  while  the  cones  are 
long  and  narrow.  The  other  layers  are  reduced  to  a minimum  at  the  fovea 
centralis,  but  at  the  margins  of  the  fovea  they  are  thicker.  There  are  a large 
number  of  ganglionic  bipolar  cells.  - 

24.  V.S.  Entrance  of  Optic  Nerve. — Make  from  an  eyeball  stained  in 
bulk  in  borax-carmine  a section  longitudinally  through  the  optic  nerve  to 
include  its  entrance  into  the  eyeball.  Observe  the  lamina  crihrosa^  ^.e.,  the 
felt- work  of  fibrous  tissue  of  the  sclerotic,  perforated  by  the  fibres  of  the  optic 
nerve. 

25.  Eye  of  Triton  Cristatus. — Excise  one  eyeball,  pin  it  to  the  under  surface 
of  a cork,  and  fix  the  cork  in  a glass  thimble  containing  a little  osmic  acid. 
In  ten  minutes  or  so  the  retina  is  “fixed,”  owing  to  the  tenuity  of  the 
sclerotic.  Divide  the  eyeball  into  two  by  a cut  at  the  equator,  place  the 
posterior  half  in  dilute  alcohol  (6  to  10  hours),  and  then  in  picro-carmine  for 

^ Archiv /.  mik.  Anal.,  xxxviii.  p.  317,  1891. 


EAR — NOSE. 


371 


xxxiil] 

the  same  period.  Fix  it  finally  in  osmic  acid,  embed  and  cut  it  in  soft  paraffin. 
The  retina  of  this  animal  is  particularly  serviceable,  because  its  structural 
elements  are  so  large. 

26.  Lachrymal  gland  is  treated  precisely  in  the  same  way  as  the  serous 
salivai-y  glands  (Lesson  XXIII. ). 


LESSOR  XXXIIL 

BAR— NOSE. 

EAR. 

1.  Membrana  Tympani  (H). — Fix  it  in  osmic  acid  and  mount 
in  Farrant^s  solution.  Observe  the  radiate  yellow-looking  fibres, 
and  also  fibres  disposed  circularly,  the  latter  best  developed  near 
the  periphery  of  the  membrane.  The  thin  epithelial  coverings  on 
the  two  surfaces  of  the  membrane  can  he  seen,  and  so  can  a few 
fine  blood-vessels. 

2.  Ceruminous  Glands  of  the  Meatus. — Harden  in  absolute 
alcohol  a portion  of  skin  from  the  external  auditory  meatus,  pre- 
ferably from  a new-born  infant.  Make  rather  thick  sections,  stain 
in  haematoxylin  and  mount  in  balsam.  Observe  the  ceruminous 
glands,  which  in  some  respects  are  like  the  sweat-glands.  They 
have  a narrow  duct  and  a coil,  the  latter  lined  by  a single  layer  of 
cubical  cells,  and  the  former  by  several  layers  of  cells,  as  in  a sweat- 
gland.  In  the  secretory  part  smooth  muscular  fibres  lie  between 
the  epithelium  and  the  basement  membrane.  The  lumen  of  the 
secretory  part  is  very  wide,  and  the  lining  cells  have  a clear  cuti- 
cular  disc,  and  contain  a yellowish  pigment  and  fatty  granules. 

THE  COCHLEA. 

Methods. — (i.)  In  a freshly-killed  rabbit  or  guinea-pig,  inject, 
by  means  of  a hypodermic  syringe,  osmic  acid  (i  p.  c.)  through  the 
membrana  tympani  into  the  middle  ear.  Cut  away  the  lower  jaw, 
so  as  to  expose  the  large  spherical  osseous  bulla.  Open  the  bulla, 
cut  away  its  walls,  and  expose  the  middle  ear.  On  the  inner  wall 
of  the  latter  are  seen  the  turns  of  the  cochlea.  Cut  away  the 
surrounding  parts  from  the  osseous  cochlea,  and  place  the  latter  in 
Muller’s  fluid  (a  week),  after  opening  one  of  the  turns,  to  allow  the 
hardening  fluid  to  penetrate.  It  is  better  to  do  this  under  fluid,  to 
prevent  air  entering,  as  the  perilymph  escapes.  Decalcify  in  chromic 


372 


PRACTICAL  HISTOLOGY. 


[XXXIII. 


and  nitric  acid  fluid,  and  harden  in  the  dark  in  alcohol.  Stain  the 
softened  cochlea  in  hulk  in  borax-carmine.  Embed  in  paraffin,  and 
cut  sections  from  base  to  apex  of  the  coiled  tube.  Fix  on  a slide, 
and  mount  in  balsam.  Use  the  other  decalcified  cochlea,  stained  as 
above,  and  cut  it  in  celloidin. 

(ii.)  The  following  is  a better  method  (Kanvier),  and  preserves 
the  finer  structures  in  situ.  Open  the  bulla  of  a guinea-pig,  and 
cut  out  the  inner  ear.  Make  an  opening  or  two  in  the  turns  of  the 
cochlear  tube,  and  place  it  in  1-2  cc.  of  i per  cent,  gold  chloride. 
Add  to  the  gold  solution  from  time  to  time  a few  cc.  of  the  follow- 
ing mixture  : — i per  cent,  gold  chloride  and  a fourth  part  of  formic 
acid,  boil,  and  allow  the  mixture  to  cool.  Leave  the  cochlea  in  the 
gold  (20-30  minutes),  remove  it,  wash,  and  expose  it  to  light  in 


Fig.  342.— V.S.  Cochlea  of  Guinea-Pig.  F.  Scala  vestibuli ; T.  Scala  tympani ; VT.  There 
the  two  communicate  ; C.  Cochlear  canal  or  scala  media ; R.  Reissner’s  membrane  ; 
t.  Membrana  tectoria. 

water  slightly  acidulated  with  acetic  acid  (1-2  drops  to  20  cc.  water). 
After  2-3  days  harden  it  in  alcohol  and  decalcify  it  in  picric  acid. 
Embed  in  gum  and  harden  in  alcohol,  or  embed  it  in  celloidin 
and  make  sections  parallel  to  the  axis  of  the  cochlea. 

(iii.)  After  removing  the  inner  ear,  open  its  osseous  canal  under 
osmic  acid  .2  per  cent,  and  leave  it  in  this  fluid  for  10-12  hours. 
Wash  in  water  and  decalcify  in  chromic  acid,  2 per  1000. 

(iv. ) After  treating  the  cochlea  with  osmic  acid  and  hardening  it 
in  alcohol,  the  following  decalcifying  fluid  may  be  used  : — 1 cc.  of 
a I per  cent,  solution  of  chloride  of  palladium,  10  cc.  of  hydrochloric 
acid,  and  1000  cc.  of  water. 

3.  V.S.  Cochlea,  parallel  to  its  axis. 

(a.)  (L)  The  turns  of  the  cochlear  tube  cut  across  (fig.  342),  each 
tube  divided  by  a transverse  partition — the  lamina  spiralis — into 


xxxiil] 


THE  COCHLEA. 


373 


an  upper — scala  vestibnli — and  a lower  compartment— scala  tym- 
pani ; the  central  column  or  mcdiolus ; the  last  is  osseous  and 
contains  channels  for  nerves  and  vessels.  The  lamina  spiralis  is 
partly  osseous,  viz.,  that  part  next  the  modiolus,  and  partly  mem- 
branous, viz.,  that  part  next  the  wall  of  the  cochlea;  the  latter 
part  is  the  membrana  basilaris.  The  osseous  portion  terminates 
in  a crest — the  crista  spiralis — and  its  free  end  is  scooped  out  into 
a groove — sulcus  spiralis. 

(h.)  From  the  u])per  surface  of  the  lamina  spiralis  ossea  there 
runs  to  the  wall  of  the  cochlea  a thin  membrane — membrane  of 


Fig.  343.— Scheme  of  the  Canalis  Cochlearis  and  the  Organ  of  Corti.  N.  Cochlear  nerve  ; 
i.  Inner  row,  and  p.  Outer  rows  of  hair-cells  ; n.  Nerve-fibrils  terminating  in  p ; 
a.  Supporting  cells  for  p;  d Cells  in  the  sulcus  spiralis  ; G and  H.  Epithelial  cells  ; 
0.  Membrana  reticularis  ; z.  Inner,  and  y.  Outer  Rod  of  Corti. 


Reissner — which  completely  shuts  off  a small  three-sided  cavity 
from  the  scala  vestibuli.  This  is  the  ductus  cochlearis,  canalis 
cochlearis,  or  scala  media.  It  is  bounded  by  the  wall  of  the  cochlea, 
the  membrane  of  Eeissner — and  its  base  is  formed  by  the  outer 
part  of  the  osseous  and  the  whole  of  the  membranous  spiral  lamina. 
The  latter  is  called  the  basilar  membrane.  This  cavity  contains 
a fluid,  the  endolymph,  and  into  it  pass  the  cochlear  branches  of  the 
auditory  nerve  to  end  in  a complex  structure — organ  of  Corti — 
which  rests  on  the  basilar  membrane. 

(c.)  'Note  in  the  scala  media  a membrane — membrana  tectoria 


374 


PRACTICAL  HISTOLOGY. 


[XXXIII. 


— which  arises  from  the  lamina  spiralis  ossea,  and  spreads  over  so 
as  partially  to  cover  the  organ  of  Corti.  The  organ  of  Corti  con- 
sists of  two  rows  of  pillars — inner  and  outer — the  rods  of  Corti — 
which  meet  above,  forming  arches,  and  leave  a three-sided  tunnel 
between  them.  Internal  and  external  to  these  are  rows  of  cells, 
some  of  them  provided  with  fine  bristles — hair-cells — constituting 
the  cells  of  Corti  and  Deiters.  Some  of  these  cells  are  the  actual 
terminal  end-organs  of  the  cochlear  nerve,  and  others  are  susten- 
tacular  in  function.  The  scala  vestibuli  and  scala  tympani  com- 
municate at  the  apex  of  the  cochlea,  as  shown  in  fig.  342,  Y.T. 

(H)  Observe  the  rods  of  Corti,  the  inner  more  numerous  than 
the  outer,  and  how  the  head  of  the  one  lies  over  the  head  of  the 
other.  The  rods  vary  in  length  and  span,  in  different  parts  of 
the  cochlea.  Internal  to  the  inner  rods  is  a single  row  of  hair- 
cells,  and  external  to  the  outer  rods  are  several  rows  of  hair-cells. 
Between  the  latter  are  supporting  cells,  and  beyond  them  are 
columnar  epithelial  cells  (fig.  343).  The  rods  and  hair-cells  are 
covered  by  a special  membrane,  seen  in  section,  which  is  perforated 
by  the  upper  ends  of  the  hair-cells — membrana  reticularis — the 
basilar  membrane  terminating  towards  the  wall  of  the  cochlea  in  a 
fibrous  expansion,  the  spiral  ligament. 

The  student  must  give  considerable  time  and  attention  to  the 
subject  if  he  wishes  to  get  preparations  showing  the  structure  of  all 
the  parts.  It  is  difficult  to  keep  the  parts  from  falling  asunder,  and 
if  the  ear  be  not  properly  decalcified,  bubbles  of  gas  are  discharged 
within  the  cochlea,  which  rupture  the  finer  parts  ; hence  arises  the 
necessity  for  opening  the  cochlea  and  fixing  the  parts  with  a “ fix- 
ing medium  previous  to  decalcifying  it. 

4.  Semicircular  Canals. — Fix  the  membranous  semicircular 
canals,  and  their  ampullae,  of  a skate  in  Flemming^s  or  Fobs  fluid. 
Harden  in  alcohol.  In  the  ampullae  are  the  terminations  of  the  audi- 
tory nerve  in  the  crista  acustica.  Make  T.S.  of  the  canals  and  Y.S. 
of  the  crista  acustica ; in  the  latter  case  take  care  to  include  the 
entrance  of  the  nerve-fibres.  The  ^preparation  of  suitable  specimens 
to  stain  the  termination  of  the  nerves  in  the  crista  acustica  presents 
very  considerable  difficulties.  The  student  may  have  to  repeat  the 
process  several  times  if  he  wishes  to  get  typical  specimens. 


THE  NOSE. 

5.  Olfactory  Mucous  Membrane. — Divide  longitudinally  the 
head  of  a freshly-killed  rabbit.  Place  small  pieces  of  the  olfactory 
mucous  membrane — readily  recognised  by  its  brownish  colour — 
in  dilute  alcohol  (2  hours),  and  then  in  i per  cent,  osmic  acid 


xxxiil] 


THE  NOSE. 


375 


(24  hours).  Harden  in  alcohol,  make  Y.S.,  stain  with  haematoxylin, 
and  mount  in  balsam. 

(a.)  (L)  Observe  on  the  surface  the  row  of  columnar  epithelial 
cells  with  oval  nuclei,  and  under  these  numerous  rows  of  cells  with 
spherical  nuclei.  At  the  base  of  the  latter  a row  of  more  granular- 
looking  basal  cells  (fig.  344).  The  basis  of  the  mucous  membrane 
composed  of  connective  tissue  with  sections  of  numerous  glands — 
Bowman’s  Glands.  Sections  of  blood-vessels  and  branches  of  the 
non-medullated  olfactory  nerve  may  also  be  seen. 

6.  Isolated  Olfactory  Cells. — Place  small  pieces  of  the  olfactory 


Fig.  344. — V.S.  Olfactory  Region  (Rabbit),  s.  Disc 
on  cells ; zo  and  zr.  Zones  of  oval  and  round 
nuclei ; b.  Basal  cells ; dr.  Part  of  Bowman’s 
glands  ; n.  Branch  of  olfactory  nerve. 


Fig.  Q45.  — Olfactory 
Cells.  jV.  Human; 
n.  Frog  ; E.  Sup* 
porting  cell. 


mucous  membrane  in  dilute  alcohol  (24  hours).  Fix  the  membrane 
in  I per  cent,  osmic  acid  (5  minutes).  Stain  the  pieces  in  bulk  in 
picro-carmine  (24  hours).  Scrape  off  a little  of  the  stained  and 
softened  epithelial  covering  and  mount  it  in  glycerine. 

(H)  («.)  Observe  the  olfactory  cells  as  very  narrow,  cylindrical, 
elongated  cells  with  a large  spherical  nucleus,  much  broader  than 
the  body  of  the  cell.  The  free  surface  carries  fine  cilia,  but  they 
are  apt  to  be  displaced  in  the  process  of  teasing  the  tissue  (fig.  345). 

(b.)  Supporting  cells,  not  unlike  columnar  epithelium,  but  they 
have  a large  oval  nucleus  (fig.  343,  E).  They  have  a long  central 


PRA.CTICAL  HISTOLOGY. 


376 


[XXXIII. 


process.  The  free  surface  of  the  cell  is  covered  hy  a clear  hem,  hut 
its  exact  constitution  and  significance  are  unknown. 

(c.)  There  may  also  be  found  large  granular  polygonal  cells 
derived  from  Bowman’s  glands — their  acini  and  ducts. 


ADDITIONAL  EXERCISES. 

Olfactory  Bulb. 

1.  This  is  a very  complicated  structure,  and  has  been  investigated  recently 
by  Golgi  and  Ih  y Cayal.  The  bulb  of  a new-born  animal  is  hardened  by 
Golgi’s  rapid  method  and  sections  made.  It  contains  a layer  of  white  nerve- 
tibres,  and  under  this  large  nerve-cells — mitral  cells — and  one  remarkable 
arrangement  known  as  olfactory  glomeruli,  which  are  nests  of  fibrils,  formed 
partly  by  the  terminal  arborisations  of  the  processes  of  mitral  cells  and  by  non- 
medullated  nerve-fibres  of  the  olfactory  nerve,  which  are  passing  onwards  to 
terminate  in  the  olfactory  epithelium. 

2.  Olfactory  Region  by  Golgi’s  Method. — The  mucous  membrane  of  the 
olfactory  region  is  placed  for  7 days  in  Golgi’s  osmico -bichromate  fluid,  then  in 
silver  nitrate,  and  liardened  in  alcohol.  One  often  fails,  but  if  a successful 
preparation  be  obtained  the  ends  of  the  olfactory  nerve  are  seen  passing  into 
the  olfactory  cells. 

3.  T.S.  Nose. — Harden  the  whole  nose  of  a mouse  in  Miiller’s  fluid,  stain  in 
bulk  in  borax-carmine,  and  make  T.S.  across  the  whole  organ  to  show  its 
walls,  septum,  turbinated  bones,  respiratory  and  olfactory  regions.  In  such 
animals  as  possess  a well-developed  organ  of  Jacobson  study  it.  In  it  also, 
if  a young  animal,  will  be  found  beautiful  sections  of  developing  tooth. 


LESSON  XXXIV. 

TERMINATION  OF  NERVES  IN  SKIN  AND  SOME 
MUCOUS  MEMBRANES. 

Sensory  nerve-fibres  terminate  in  the  skin  and  mucous  mem- 
branes in  three  ways  : — 

(I.)  Free  nerve-endings,  i.e.,  by  intra-epidermic  fibrils. 

(II.)  Ill  special  terminal  corpuscles. 

(III.)  In  neuro-epithelial  cells,  ^.e.,  specially  modified  epithe- 
lial cells,  as  the  rods  and  cones  of  the  retina,  the 
auditory  hairs  in  the  ear,  the  inner  cells  of  taste-buds 
and  olfactory  cells. 

I.  Free  nerve-endings  occur  especially  in  stratified  epithelium, 
e.g.y  ill  the  skin,  mouth,  and  cornea  (Lesson  XXXII.).  The  nerve- 
fibres  lose  their  myelin  and  primitive  sheath,  and  the  axis-cylinder 


XXXIV.] 


NERVES  OF  SKIN. 


377 


splits  up  into  fine  fibrils,  wliicli  penetrate  into  the  epithelial  layer 
and  run  between  the  epithelial  cells,  and  sometimes  anastomose 
with  each  other,  to  end  in  free  points  which  do  not  form  connec- 
tions with  any  structure  in  the  epidermis.  In  the  skin  these 
fibrils  are  confined  to  the  rete  Maipighii,  and  do  not  reach  the 
stratum  granulosum. 

I.  V.S.  Skin  {gold). — Use  boiled  gold  chloride  and  formic  acid 
(p.  372).  Take  very  small  pieces  (2  mm.  cubes)  of  the  palmar 
skin  of  the  fingers  or  toes,  preferably  from  a new-born  child  or  a 
young  infant;  cut  away  all  the  adipose  tissue,  and  place  the  pieces 
in  the  gold  mixture  for  at  least  one  hour.  Then  place  the  tissue  in 
slightly  acidulated  water,  and  expose  to  sunlight  until  the  gold  is 
reduced.  Harden  in  alcohol,  and  mount  the  sections  in  formic 
glycerine. 

(H)  Observe  the  blackened  fine  nerve-fibrils.  Some  of  them 
beaded,  running  between  the  epithelial  cells  (fig.  336). 

II.  Terminal  Corpuscles. — They  are  very  varied  in  their  form, 
and  include  the  following  : — 

A.  Simple  tactile  cells. 

B.  Compound  tactile  cells. 

C.  End-hulhs  loitJi  many  modifications. 

D.  Touch-corpuscles. 

A.  Simple  Tactile  Cells— V.S.  Skin  {gold)  (H).— Treat  very 
small  pieces  of  the  human  skin 
(volar  surface  of  finger  or  toe) 
or  the  snout  of  a pig  by  the 
boiled  gold  formic  acid  method. 

{a.)  Observe  a nerve-fibre 
(fig.  346,  n)  passing  towards 
and  entering  the  epidermis, 
where  it  is  non-medullated  and 
splits  into  fibrils,  which  ter- 
minate in  oval,  nucleated,  tactile 
discs  or  menisci  (m),  each  of 
which  lies  under  a tactile  cell 
{a).  The  cells  are  6-12  /x  in 

length,  and  lie  in  the  deeper  Fig.  346.— Tactile  Cells,  Snont  of  P:g.  n.  Nerve- 
, p j 1 *1  • fibre;  a.  Tactile  cell ; m.  Tactile  disc, 

part  01  the  epidermis. 

B.  Compound  Tactile  Cells,  called  also  G-randry’s  and  Merkel’s 
Corpuscles,  consist  of  two  or  more  cells  (15  //.  x 50  /x)  piled  one  on 
the  other ; between  each  two  cells  is  a disc  or  plate — tactile  disc — 
which  is  connected  with  the  axis-cylinder  of  a nerve.  The  cells 
have  a large  pale  nucleus,  and  the  whole  is  surrounded  by  a fibrous 
capsule.  The  nerve  loses  its  myelin  after  it  passes  into  the  organ, 


3/8  PRACTICAL  HISTOLOGY.  [XXXIV. 

and  its  sheath  becomes  continuous  with  the  fibrous  capsule.  So 

far  these  structures  have  been 
found  only  in  the  bill  and  tongue 
of  birds,  e.g.^  duck.  They  lie  in 
the  coriurn,  close  under  the  epi- 
dermis (fig.  347). 

3.  T.S.  Tongue  or  Bill  of 
Duck  (Grandry’s  Corpuscles) 
(L  and  H).  — Harden  in  i per 
cent,  osmic  acid  small  pieces  of 
the  marginal  part  of  the  tongue 
or  the  sieve-like  structure  on 
the  edges  of  the  mandibles  of  a 
duck.  Treat  other  small  pieces  by  the  boiled  gold  chloride  method. 

(a.)  Observe  the  tactile  cells  and  discs  as  in  fig.  347. 

C.  End-bulbs  are  small,  oval,  or  cylindrical  bodies  of  various 
shapes.  A nerve-fibre  enters  one  pole,  and  as  it  does  so  it  loses  its 
myelin  and  terminates  in  a softer  granular  inner  core.  The  bulb — 

which  may  be  cylindrical  in  shape 
— consists  of  a tough  fibrous  cap- 
sule continuous  with  the  sheath  of 
the  nerve  ; and  a softer  inner  core 
in  which  the  axis-cylinder  ter- 
minates. Such  bodies  are  found 
in  the  connective  tissue  of  the 
mucous  membrane  of  the  mouth 
and  conjunctiva.  It  is  not  difficult 
to  isolate  them  from  the  conjunc- 
tiva of  a calf  (fig.  348).  They  lie 
in  the  sub  - epithelial  connective 
tissue.  Certain  special  forms  of 
them  occur  in  the  genital  organs, 
Fia.  348.— End-Bulb  from  Human  Con-  c.y.,  clitoris,  and  in  connection  with 

junctiva.  a.  Nucleated  capsule;  h.  4^,*, A c?nppial  form  Lnnwn 

Core;  c and  d.  Nerve  terminating  jCiniS.  special  lOrm  IS  Known 

in  d,  as  Pacinian  corpuscles. 

4.  Pacinian  Corpuscles  {Cat), 
— These  are  elliptical  transparent  bodies  (2-3  mm.  long  and  i mm. 
thick),  readily  found  in  the  meso-colon  and  mesentery  of  a cat. 
They  consist  of  many  concentric  fibrous  laminae,  arranged  like  the 
coats  of  an  onion,  surrounding  a central  core,  which  is  continuous 
with  the  axis-cylinder  of  a nerve.  The  lamellae  are  lined  on  both 
surfaces  by  endothelium,  which  can  be  stained  with  silver  nitrate 
(fig.  350).  The  capsules  are  closer  together  near  the  centre  than 
they  are  at  the  periphery.  Examine  one  fresh  in  normal  saline, 
and  “fix’^  another  with  osmic  acid  and  mount  it  in  Farrant^s 


Fig.  347.— Tactile  Corpuscles,  Bill  of  Duck, 
A.  With  three,  and  B.  With  two  tactile 
cells.  The  axis-cylinder  terminates  in  the 
tactile  disc  (black). 


XXXIV.] 


NERVES  OF  SKIN. 


379 


solution.  A medullated  nerve-fibre  surrounded  by  perineurium 
passes  to  each  corpuscle.  The  lamellae  of  the  perineurium  become 


Fig.  349.— Pacini’s  Corpuscle,  from  Mesen- 
tery of  Cat.  c.  Capsules  ; d.  Endothe- 
lial lining  separating  the  latter;  n. 
Nerve ; /.  Funicular  sheath  of  nerve  ; 
m.  Central  mass ; n'.  Terminal  fibre  ; 
and  a.  Where  it  splits  up  into  finer 
fibrils.  Examined  in  fresh  normal 
saline. 


Fig.  350. — Endothelium  of 
Lamellae  of  a Pacinian 
Corpuscle.  Silver  nitrate. 


continuous  with  the  lamellae  of  the  corpuscle,  and  the  nerve — still 
provided  with  its  myelin  and  sur- 
rounded by  its  endoneurium  — pierces 
the  lamellae  and  reaches  the  central 
part  of  the  corpuscle.  The  endoneurium 
seems  to  form  a soft  core  round  the  axis- 
cylinder,  which  usually  sj)lits  up  at  the 
further  end  into  a tuft  of  fibrils  (fig. 

349)- 

5.  T.S.  Pacinian  Corpuscles. — They 
lie  in  the  subcutaneous  tissue.  In  a 
Y.S.  of  foetal,  skin  from  the  palmar 
surface  of  a digit  (Lesson  XXIX.  5)  it 
is  easy  to  find  these  bodies  cut  obliquely 
or  transversely.  Fig.  351  shows  their  appearance  when  cut  trans- 
versely. Harden  the  skin  in  alcohol  or  osmic  acid.  Stain  in  picro- 
carmine  or  hsematoxylin. 

6.  Herbst^s  Corpuscles  (H)  are  modified  Pacinian  corpuscles, 
occurring  in  the  mucous  membrane  of  the  tongue  of  the  duck.  They 
lie  in  the  corium,  just  under  the  epidermis.  Harden  the  tongue  or 
bill  as  for  tactile  corpuscles  (p.  378),  or  use  absolute  alcohol,  and 
stain  the  tissue  in  bulk  with  borax-carmine.  Mount  in  balsam. 


Fig. 


51.-— T.S.  Pacinian  Corpuscle, 
Fuutal  Skin. 


38o 


PRACTICAL  HISTOLOGY. 


[XXXIV. 


(a.)  Observe  the  same  general  arrangement  as  in  Pacinian  cor- 
puscles. These  bodies,  however,  are  more 
elongated,  and  the  axis-cylinder  . in  the 
centre  is  bordered  by  a row  of  nuclei. 

D.  Touch-Corpuscles  of  Wagner  or 
Meissner.  — These  are  elliptical  bodies 
(40-1 50  /X  long  and  30-60  fx  broad),  which 
occur  in  the  papillae  of  the  skin,  especially 
in  the  volar  side  of  the  fingers  and  toes. 
A nerve-fibre  passes  to  each  corpuscle, 
which  has  a fibrous  sheath.  Owing  to  the 
way  in  which  the  divisions  of  the  nerve 
twist  round  the  corpuscle,  they  have  an 
irregular  transversely  striated  appearance. 
The  nerve-fibre  enters  the  corpuscle,  and 
its  branches  take  a coiled  course  round  the 
corpuscle. 

7.  V.S.  Skin  (Touch-Corpuscles). — (i.) 

Make  vertical  sections  of  skin  (palmar 
surface  of  digit),  harden  in  alcohol,  stain 
them  in  hsematoxylin  or  safranin,  and 
mount  in  balsam  (fig.  352). 

(ii.)  Treat  skin  by  the  boiled  gold 
chloride  method  to  see  the  terminal 
branches  and  distribution  of  the  nerve 
within  the  corpuscle  (fig.  353).  In  some 
situations  the  corpuscles  are  compound. 

The  following  Table  shows  the  modes 
of  termination  of  nerve-fibres  in  sensory 
surfaces : — 


A 

Fig.  352.— Y.S.  Skin,  Palm  of 
Hand.  h.  Papilla  of  cutis ; 
d.  Nerve-fibre  in  touch-cor- 
puscle ; e,f.  Nerve-fibre  in 
touch-corpuscle ; g.  Cells  of 
Malpighian  layer.  Alcohol 
and  safranin. 


I. 

II. 

III. 

IV. 

Free  nerve -end- 
ings (cornea, 
skin). 

Simple  tactile- 
cells  (human 
skin). 

Compound  tac- 
tile-cells 
(birds,  e.g., 
duck’s  bill 
and  tongue  . 

Cylindrical  end-bulbs 
(conjunctiva). 

Herbst’s  corpuscles 
(tongue  of  duck). 

Pacinian  or  Vater’s 
corpuscles(deep  part 
of  skin,  mesentery 
of  cat,  and  near 
joints). 

Genital  corpuscles 

(clitoris). 

Wagner’s  touch -cor- 
puscles (papillae  of 
skin). 

Neuro  - epithe- 
lium (retina, 
internal  ear, 
olfactory  re- 
gion of  nose, 
and  organ  of 
taste). 

XXXIV.] 


NEKVES  OF  SKIN. 


381 


ADDITIONAL  EXERCISES. 


8.  Tactile  Hairs  and  Nerves  to  Hair-Follicles. — These  are  to  be  studied 
the  boiled  gold  chloride  method.^ 


9.  Organ  of  Eimer. — Prepare  the  nose  of  the  mole  by  the  gold  chloride 
method.  In  sections  study  the  termination  of  the  nerve- fibrils  in  the  structure 
known  as  the  organ  of  Eimer  (fig.  354). 


LESSON  XXXV. 

THE  TESTIS. 

The  framework  of  the  testis  (fig.  355)  consists  of  a strong,  tough, 
fibrous  capsule — the  tunica  albuginea — which  is  covered  externally 
by  endothelium  reflected  from  the  serous  membrane — the  tunica 
vaginalis.  At  the  back  part  of  the  organ,  the  tunica  albuginea  is 
prolonged  for  some  distance  (8  mm.)  into  the  gland  to  form  the 
corpus  Highmori  or  mediastinum  testis.  From  the  under  surface 
of  the  tunica  albuginea  septa  or  trabeculae  pass  towards  the  corpus 
Highmori,  and  thus  subdivide  the  gland  into  compartments  or  lobules 
(about  120  in  number),  with  their  bases  directed  outwards  and 
1 Hoggan,  Jour,  of  Anat.  and  Phys.,  1892. 


382 


PRACTICAL  HISTOLOGY. 


[xxxv. 


Septa. 

Tunica  albuginea. 

Cavity  of  tunica 
vaginalis. 

Outer  layer  of  tunica 
vaginalis. 

Vas  deferens  cut. 
Spermatic  vein. 


their  apices  towards  the  corpus  Highmori.  The  tunica  albuginea 
consists  of  dense  fibrous  tissue,  but  it  is  of  looser  texture  internally, 
where  it  has  numerous  vessels  and  lymphatics.  This  inner  layer  is 

sometimes  called  the  tunica 
vasculosa,  and  is^  continuous 
with  the  septa.  Each  lobule 
contains  2-8  seminal  tubules. 
These  convoluted  tubules  (800) 
begin  by  a blind  extremity, 
and  are  of  considerable  length 
(30-50  cm.)  and  of  nearly 
uniform  diameter  (.3  mm.  or 
140  /x).  The  tubules  of  any 
one  compartment  unite  at 
acute  angles  to  form  a small 
number  of  much  narrower 
straight  tubules  (20-25  — 

tubuli  recti— which  pass  into 
the  mediastinum  and  there  form  a network  of  inter-communicating 
tubules  of  irregular  diameter  (24-180  /x) — rete  testis  (fig.  356). 

From  this  proceed  12-15  wider 
ducts — vasa  efferentia — which 
are  straight  at  first,  hut  some 
become  convoluted  and  form  a 
series  of  conical  eminences — 
coni  vasculosi — which  together 
form  the  head  of  the  epididy- 
mis. The  epididymis,  consist- 
ing of  a single  convoluted  tube 
(600  cm.,  or  20  feet  long),  is 
continued  into  a thick-walled 
muscular  tube  — vas  deferens 
(60  cm.,  or  2 feet  long) — which 
conducts  the  secretion  to  the 
urethra,  and  is,  in  fact,  the 
excretory  duct  of  the  testis. 

The  seminiferous  tubules 
consist  of  a thick  membrane 
composed  of  flattened  nucleated 
cells,  arranged  like  membranes 
and  lined  internally  by  a base- 
ment membrane.  The  latter  is 
lined  by  several  rows  of 
in  appearance,  according  to 
In  the  resting  condition. 


Arteries.  Corpus  Epididy- 
Highmori  mis. 

Fig.  355.— T.S.  of  Testis. 


End  of  con- 
voluted tube. 


Rete  Testis. 


Fig.  356.— Convoluted  and  Straight  Tube  of 
Testis. 


cubical  cells,  which  differ  much 
the  functional  activity  of  the  organ. 


XXXV.] 


THE  TESTIS. 


383 


the  tubes  are  lined  by  several  layers  of  more  or  less  cubical  cells, 
whose  nuclei  stain  more  or  less  deeply  with  staining  reagents.  In 
an  active  gland  in  some  tubules  some  of  the  clear  cubical  cells  of 
the  outer  row  of  cells — lining  epithelium — contain  nuclei  under- 
going mitotic  division.  The  rows  of  cubical  cells  internal  to  these 
have  a radiate  arrangement,  and  are  separated  into  groups  by  larger 
structures,  sometimes  called  spermatoblasts,  and  by  other  observers 
sustentacular  cells,  which  grow  up  between  the  smaller  cells, 
and  at  their  upper  ends  are  connected  with  the  spermatozoa.  Inside 
the  layer  of  lining  epithelial  cells  are  several  rows  of  larger  cells — 
spermatogenic  cells — with  nuclei  showing  different  stages  of 
mitosis.  I^ext  the  lumen,  which  is  always  well  defined,  in  some  of 
the  tubules  are  spermatozoa  in  different  stages  of  development.  In 
others,  however,  not  so  far  advanced,  the  inner  row  of  cells  consists 
of  small  protoplasmic  cells — the  true  spermatoblasts — as  from  these 
cells  the  spermatozoa  are  developed.  The  developing  spermatozoa 
rest  by  means  of  their  heads  on  the  sustentacular  cells.  The  sper- 
matozoa are  arranged  in  tufts  or  groups,  with  their  tails  towards 
the  lumen.  The  spermatozoa  gradually  develop  from  the  true 
spermatoblasts  and  pass  towards  the  lumen,  and  as  they  are  set 
free  new  spermatoblasts  are  formed  by  the  mitotic  division  of  the 
spermatogenic  cells. 

The  straight  tubules  are  lined  by  a single  layer  of  flattened 
epithelium ; the  rete  testis  has  no  basement  membrane,  but  it 
also  is  lined  by  a single  layer  of  flattened  epithelium.  The  vasa 
efferentia  and  the  tube  of  the  epididymis  have  smooth  muscular 
fibres  in  their  walls,  and  are  lined  by  columnar  ciliated  epithelium, 
the  cilia  being  very  long. 

The  interstitial  tissue  of  the  testis  between  the  tubules  is  very 
loose  in  texture  and  laminated,  and  has  numerous  lymphatic  slits. 
In  some  animals  (rat,  boar)  are  numerous  polyhedral,  nucleated, 
sometimes  pigmented,  cells,  the  remains  of  the  cells  of  the  Wolffian 
bodies. 

The  vas  deferens  consists  of  a fibrous  coat  investing  an  outer 
thick  layer  of  smooth  muscle  arranged  longitudini^lly ; inside  this 
is  a thick  layer  of  smooth  muscle  arranged  circularly ; inside  this 
again  is  a submucous  coat  of  connective  tissue.  In  some  parts  of  the 
tube  there  is  a layer  of  smooth  muscle  arranged  longitudinally  just 
internal  to  the  circular  coat.  Then  follows  the  mucosa  lined  by 
columnar  non-ciliated  epithelium. 

Methods. — (i.)  Harden  the  fresh  testis  in  Miiller^s  fluid  (2 
weeks).  If  it  be  large,  cut  it  into  small  pieces.  The  capsule  exerts 
considerable  pressure  on  the  gland  substance,  so  that  when  the  testis 
is  cut  into,  the  latter  projects  somewhat.  Complete  the  hardening 
in  alcohol.  Corrosive  sublimate  is  a good  hardening  reagent.  Stain 


3^4 


PRACTICAL  HISTOLOGY. 


[xxxv. 


sections  in  h?ematoxylin  or  picro-carmine,  and  mount  in  balsam. 
The  hardened  testis  of  a small  animal,  e.g,^  rat,  should  also  be 
stained  in  bulk  in  borax-carmine  or  Kleinenberg’s  logwood,  and  cut 
in  paraffin,  so  as  to  include  the  body  of  the  testis  and  the  epididymis. 
Mount  in  balsam. 

(ii.)  Harden  very  small  pieces  of  the  testis  of  a freshly-killed 
rat  or  mouse  in  Flemming’s  mixture  and  stain  the  sections  in 
safranin.  A very  small  testis  is  taken,  as  Flemming’s  fluid  only 
penetrates  a few  mm.  into  the  tissue.  This  is  for  the  study  of 
spermatogenesis. 

(iii.)  Harden  the  testis  of  a frog  in  absolute  alcohol  and  stain 
it  in  bulk  in  Kleinenberg’s  logwood. 

(iv.)  Harden  the  testis  of  the  dogfish  in  Flemming’s  fluid  and 
stain  the  sections  in  hsematoxylin  or  safranin.  This,  as  shown  by 
Swaen,  is  an  excellent  object  for  the  study  of  spermatogenesis. 

(v.)  Harden  the  testis  of  a mouse,  salamander,  or  frog  in  the 
following  fluid  ^ in  which  platinic  chloride  is  substituted  for  the 
chromic  acid  in  Flemming’s  chromo-aceto-osmic  acid  mixture : — 
Hermann’s  Fluid, 

Platinic  chloride  (i  per  cent.)  . 15  parts. 

Osmic  acid  (2  per  cent.)  . . 4 

Glacial  acetic  acid  . . . i part. 

The  above  is  used  for  the  tissues  of  mammals ; but  if  a salamander 
testis  is  used,  then  take  only  2 parts  of  the  osmic  acid. 

After  2-3  clays  the  details  of  the  cells  are  better  brought  out 
than  with  Flemming’s  solution.  Complete  the  hardening  in  alcohol. 
Make  sections  in  paraffin,  fix  them  on  a slide  with  albumin  fixative, 
and  stain  for  24-48  hours  in  the  following  safranin  fluid  : — 

Safranin  . . . , i gram. 

Alcohol  . . . . 10  cc. 

Water  . , , . 90  ,, 

After  staining,  wash  in  water,  acid-alcohol  and  alcohol,  but  do 
not  remove  all  the  surplus  safranin.  Then  stain  for  3-5  minutes 
in  a gentian-violet  solution.  Decolorise  the  sections  by  means  of 
Gram’s  method  (p.  105),  i.e,,  after  washing  in  alcohol,  place  them 
for  1-3  hours  in  the  following  fluid — 

Iodine  . . . . i gram. 

Potassic  iodide  . . .2  grams. 

Water  ....  300  cc. 

until  they  are  quite  black,  and  then  differentiate  them  in  alcohol. 
Mount  in  balsam. 

1.  T.S.  Testis  [Rat). — [a.)  Observe  the  thick,  tough,  fibrous 
tunica  albuginea  Avith  sections  of  large  blood-vessels  and  lymphatics 

^ Archiv  f.  mik.  Anat.j  xxxiv.  p.  58. 


XXXV.] 


THE  TESTIS. 


385 


in  its  deeper  part.  From  its  under  surface  septa  pass  into  the 
gland  at  fairly  regular  intervals,  thus  dividing  it  into  a series  of 
compartments  or  lobules.  At  the  upper  and  back  part  is  the 
fibrous  septum— corpus  Highmorianum  or  mediastinum  testis. 
Many  of  the  septa  are  connected  with  it. 

(h.)  In  the  lobules  lie  twisted  or  convoluted  tubules — the  semi- 
niferous tubules — which  converge  towards  the  mediastinum  and 
form  near  it  a number  of  straight  tubes — the  tubuli  recti — which 
in  their  turn  unite  and  form  the  rete  testis  in  the  mediastinum, 
and  from  this  proceed  the  vasa  efferentia,  which  run  to  join  the 
canal  of  the  epididymis  (fig.  356). 

{c.)  The  tubules  in  a state  of  activity  are  distinguished  from 
the  resting  ones  by  the  intensely  stained  heads  of  the  young 
spermatozoa.  An  active  tubule  is  lined  by  several  rows  of  poly- 
gonal cells,  some  of  which  are  larger  than  the  others.  Embedded 
amongst  the  cells,  near  the  lumen  of  the  tube,  are  bunches  or 
tufts  of  spermatozoa,  best  seen  in  longitudinal  sections  of  the 
tubes.  The  heads  are  directed  towards  the  wall,  and  the  tails 
towards  the  lumen  of  the  tube. 

(d.)  In  some  animals  the  interstitial  tissue  between  the  tubules 
is  chiefly  formed  by  thin  flattened  membranes  of  connective  tissue  ; 
but  in  others,  e.y.,  boar,  the  matrix  consists  of  numerous  pigmented, 
polygonal,  very  granular  cells — interstitial  cells. 

2.  Spermatogenesis  (Eat). — In  order  to  see  the  structure  of 
the  seminiferous  tubules  to  the  best  advantage,  harden  small 
pieces  of  the  testis  of  a rat,  or  a guinea-pig,  as  directed  under 
V.  and  ii.  (p.  384).  To  keep  the  parts  together,  cut  in  paraffin. 
The  best  stain  is  safranin.  Fix  a thin  T S.  of  a tubule  under  a 
high  power. 

(a.)  Observe  the  rather  thick  wall — rnemhrana  propria — of  the 
tubule,  composed  of  flattened  cells,  perhaps  of  a connective  tissue 
nature.  This  is  lined  by  three  or  more  layers  of  glandular  cells, 
which  vary  in  appearance  according  to  their  condition  of  physio- 
logical activity.  In  a state  of  rest  each  tube  is  lined  by  several 
layers  of  large  polygonal  cells  placed  one  inside  the  other. 

(h.)  In  an  active  gland^  known  by  the  evidences  of  division  of 
cells  and  by  the  development  of  spermatozoa  (fig.  357),  there  is  an 
outer  layer  of  cubical-looking  cells,  and  internal  to  it  several  layers 
of  round  or  ovoid  cells,  which  are  called  speimatogenic  cells.  In 
the  latter  may  be  seen  nuclei  undergoing  mitotic  division.  The 
lining  epithelial  cells  seem  to  divide  ; one  part  of  each  cell  passes 
into  the  second  layer  of  cells,  and  becomes  a sperm atogen  or  sper- 
matogenic  cell,  while  the  remainder  of  the  original  cell  enlarges  and 
grows  up  as  a sustentacular  cell.  The  spermatogenic  cells  divide 
and  redivide  by  mitosis,  and  yield  the  small  daughter-cells  or 
34  2 B 


386 


PRACTICAL  HISTOLOGY. 


[xxxv. 


spermatoblasts  of  the  innermost  layer.  The  latter,  arranged  in 
groups,  gradually  elongate,  and  from  them  the  groups  of  spermatozoa 
are  formed.  Each  group  rests  on  and  is  connected  with  a susten- 
tacular  cell ; hence  arose  the  old  view  that  these  sustentacular 
cells  produced  the  spermatozoa,  and  for  this  reason  they  were 
formerly  called  spermatoblasts.  The  nucleus  of  a daughter-cell 
forms  the  head  and  body  of  the  spermatozoon,  while  the  tail  is 
formed  within  the  protoplasm,  but  it  is  connected  with  the  nucleus, 
and  in  its  development  grows  out  and  forms  a cilium. 

3.  Isolated  Cells  from  the  Tubules. — Macerate  small  frag- 
ments of  the  testis  in  dilute  alcohol  10-12  hours.  Tease  a small 
fragment  in  glycerine.  Observe  the  various  forms  of  cells  iso- 
lated. 

4.  Spermatozoa  (H). — Make  a cut  into  the  epididymis  of  a testis 
removed  from  a rabbit  as  soon  as  possible  after  death.  A milky 


B 

Pig.  357. — Tubules  of  Testis  of  Rat,  showing  Spermatogenesis.  A.  Less  advanced  stage; 
£ and  C.  More  advanced  stages.  A and  R=T.S.;  C=L.S.  Flemming’s  fluid  and 
safranin,  x 300. 

juice  exudes.  Place  a little  of  this  on  a slide  and  dilute  it  with 
normal  salinOo 

(a,)  The  spermatozoa,  each  one  with  a head  and  a long  vibratile 
tail  or  cilium.  By  the  side-to-side  movement  of  the  tail  the  whole 
spermatozoon  is  moved  onwards  in  a zigzag  course. 

(b.)  Add  a drop  of  distilled  water.  The  movements  are  arrested. 
The  spermatozoa  are  slowly  killed. 

They  may  be  readily  preserved  by  smearing  a little  of  the  milky 
juice  from  the  epididymis  upon  a slide  and  allowing  it  to  dry.  It 
is  then  covered  and  sealed. 

5.  Spermatozoa  of  Newt  and  Frog  (H). — The  testis  of  a newt 
is  teased  in  a mixture  of  alcohol  and  glycerine. 

(H)  Observe  the  pointed  head,  body,  and  long  tail  of  each  sper- 
matozoon. There  is  an  intermediate  part,  which  is  best  seen  in  a 
stained  preparation.  From  it  springs  a filament  which  appears  to 
be  prolonged  as  a spiral  filament  around  the  cilium  or  tail.  The 


THE  TESTIS. 


XXXV.] 


3^7 


si)iral  filament  is  in  reality  the  optical  expression  of  a thin  membrane 
attached  in  a spiral  direction  to  the  cilium.  Mount 
the  spermatozoa  of  a frog.  The  male  frog  is  easily 
known  by  the  wart  on  his  thumb.  Open  the 
abdomen,  and  low  down  in  the  hollow  on  each 
side  of  the  vertebral  column  is  an  oval,  kidney- 
shaped, whitish  body — the  testis.  It  is  to  be 
treated  like  the  testis  of  other  animals.  At 
certain  seasons  of  the  year  no  spermatozoa  are  to  j \ 
be  found;  The  cells  are  in  a resting  phase. 

6.  Human  Spermatozoa. — Mount  in  glycerine 
some  spermatozoa  obtained  from  a spermatocele 
(fig.  358).  Observe  the  head  (k),  long  tail  (/), 
and  middle  piece  (m). 

7.  Cover-Glass  Preparations  (H). — Compress 
a small  piece  of  the  testis  of  a newt  or  frog 
between  two  cover-glasses.  Separate  them,  and 
allow  the  film  on  them  to  dry.  Stain  one  in 
methylene-blue,  and  the  other  for  twenty-four  hours 
in  Ehrlich-Biondi’s  fluid.  Dry  and  mount  in  balsam. 

(a.)  In  the  methylene-blue  specimen  only  the 
heads  of  the  spermatozoa  are  stained.  In  the 
other,  the  head  of  the  spermatozoon  is  stained 
one  colour,  while  the  tail  and  the  intermediary  or  358.  — Sperma- 

j unction  piece  is  of  a different  hue.  ^ Human. 

8.  Testis  of  Salamander.— Fix  the  testis  (in  September  or 
October)  in  Hermann’s 
fluid,  and  stain  as  directed 
at  p.  384.  The  bundles  of 
ripe  spermatozoa  are  very 
marked.  The  body  of 
each  spermatozoon  is  red- 
dish, its  head  bluish-violet, 
the  middle  piece  also  of  the 
same  colour,  while  the  tail 
is  brownish  - violet.  In 
Hermann’s  paper  figures 
will  be  found  showing  the 
remarkable  stages  of  de- 
velopment of  the  bodies 
of  the  spermatozoon  from 
the  nucleus  of  the  sperma- 
tids. numerous  mitotic 
figures  may  be  seen. 

9.  Epidiidymis  (L  and 


Blood-vessel. 


T.S.  Tubule. 


Fig=  359. — T.S.  Tubules  of  Epididymis. 


H). — Prepare  like  the  testis.  Make 


T.S.,  stain  in  hsematoxylin  and  mount  in  balsam. 


388 


PRACTICAL  HISTOLOGY. 


[xxxvi. 


(a.)  'Observe  numerous  sections  of  the  twisted  tube  of  the 
epididymis  (fig.  359);  each  tubule  has  a fibrous  wall,  and  is  lined 
by  a single  layer  of  tall,  slender,  columnar  ciliated  epithelial  cells. 
Each  cell  is  provided  with  a fringe  of  long  cilia.  The  lumen  is 
wide  and  distinct,  and  may  contain  a confused  mass  of  spermatozoa. 
At  the  bases  of  the  cells  are  sometimes  seen  small  cells,  and  in 
pigmented  animals  pigment  granules  are  not  unf requently  seen  in 
the  connective-tissue  stroma  supporting  the  tubule.  There  is  a 
fair  amount  of  connective-tissue  stroma  between  the  tubules,  and 
in  it  are  blood-vessels,  lymphatics,  and  nerves. 

10.  T.S.  Vas  Deferens. — Harden  it  like  the  epididymis,  make 
T.S.,  stain,  and  observe  the  arrangement  of  its  parts  as  described 
at  p.  383. 


LESSON  XXXYI. 

THE  OVARY— FALLOPIAN  TUBE— UTERUS. 
THE  OVARY. 

The  ovary  consists  of  a stroma,  covered  on  the  surface  with  a 
single  layer  of  short  columnar  or  germinal  epithelium.  Embedded 
in  the  stroma  are  numerous  Graafian  follicles  in  all  stages  of  ripe- 
ness. Each  Graafian  follicle  contains  one  ovum.  The  unripe 
follicles,  with  primitive  ova,  form  a superficial  layer  close  under 
the  surface,  while  the  more  mature  ova  and  follicles  lie  deeper. 
The  coverings  of  the  follicle  and  the  structure  of  a ripe  ovum  are 
given  at  p.  389. 

Methods. — Select  the  ovaries  of  small  animals,  e,^.,  mouse,  rat, 
as  they  can  be  better  “ fixed than  large  ones. 

(i.)  Harden  the  ovary  of  small  animals  in  toto  in  Muller’s  fluid 
(2  weeks),  and  then  in  alcohol.  Make  two  or  three  transverse 
cuts  in  the  human  ovary  before  hardening  it  in  the  same  fluid. 

(ii.)  Picro-sulphuric  acid,  for  a day,  is  also  a good  hardening 
reagent.  Complete  the  hardening  in  alcohol.  The  pieces  had 
better  be  stained  in  bulk  in  borax-carmine,  or  Kleinenberg’s 
logwood,  and  embedded  and  cut  in  paraffin,  or  embedded  and  cut 
in  celloidin. 

(iii.)  For  small  ovaries  use  Flemming’s  fluid,  embed  and  cut  in 
paraffin.  Stain  with  safranin. 

For  obtaining  a general  view,  the  sections  must  be  pretty  thick. 


THE  OVARY. 


XXXVI.] 


389 


Fig.  360.— T.S.  Ovary. 
Graafian  follicle  ; 
ova;  o.  Ovum; 
Stroma. 


e.  Germ  epithelium;  1.  Large 
2.  Middle-sized,  and  3.  Small 
g.  Membrana  granulosa ; s. 


otherwise  the  follicles  are  cut  into,  and  the  ova  are  apt  tb  fall  out. 
Thin  sections  must  also  he  made. 

1.  T.S.  Ovary  (fig.  360). — (a.)  (L)  The  body  of  the  ovary, 
covered  on  its  surface  by  iiii.ii.iiiiLL,.,„_ 

a single  layer  of  short, 
columnar,  nucleated  cells 
— the  germinal  epi- 
thelium. 

(6.)  The  substance  or 
stroma,  composed  of  con- 
nective tissue  with  numer- 
ous blood-vessels,  and  in 
some  places  smooth  muscu- 
lar fibres.  The  connective 
tissue  is  denser,  and  is 
arranged  in  several  layers, 
near  the  surface. 

(c.)  Lying  in  the  stroma 
— the  Graafian  follicles. 

IsTear  the  surface  is  a layer 
of  smaller  unripe  ova — 
primitive  ova  (40  /x) — 
while  the  riper  and  larger  follicles  are  situated  deeper  in  the  stroma. 
Each  follicle  contains  an  ovum,  but  if  the  section  be  thin  the  ovum 
may  drop  out  and  leave  only  the  follicle  and  its  coverings. 

(cZ.)  If  the  ovary  be  from  an  adult  animal,  a corpus  luteum  may 
be  found.  Its  appearance  will  depend  upon  whether  it  has  been 
recently  formed  or  not,  and  whether  the  animal  was  recently  preg- 
nant. If  it  is  of  some  standing,  it  is  large  and  more  or  less  spherical 
in  outline,  occupying  a considerable  part  of  the  stroma,  with  an 
umbilicus-like  centre  and  radiating  lines  of  connective  tissue.  Be- 
tween these  are  numerous  large  granular  cells.  In  a recent  one> 
there  may  be  a yellow  pigment — lutein — staining  the  scar. 

» (H)  The  germinal  epithelium,  and  under  it  the  tunica 
albu^nea,  composed  of  two  or  more  layers  of  connective-tissue 
lamellae — with  fusiform  cells — crossing  each  other.  It  gradually 
passes  into  the  stroma. 

(/)  The  Coverings  of  the  Follicle. — Select  a well-developed  or 
nearly  ripe  ovum  (.5-5  mm.  in  diameter).  Outside  is  the  theca 
folliculi,  composed  of  two  layers,  an  outer  one  fibrous — the  tunica 
fibrosa — and  inside  it  a layer  with  blood-vessels — the  tunica 
propria.  Inside  this  several  layers  of  cubical  cells,  constituting 
tlie  membrana  granulosa.  From  this  there  projects  a mass  of 
cells  at  one  part,  forming  the  cumulus  or  discus  proligerus.  The 
cells  are  continued  as  the  tunica  granulosa  around  the  ovum. 


PRACTICAL  HISTOLOGY. 


390 


[xxxvi. 


Between  the  tunica  and  membrana  granulosa  is  a space  which 
encloses  a fluid  in  the  large  follicles — the  liq^uor  foUiculi. 

{g.)  The  ovum,  composed  externally  of  a hyaline  membrane — 
the  zona  pellucida — enclosing  more  or  less  granular  cell-contents  ■ 
the  vitellus  or  yelk ; and  placed  excentrically  in  this  the  germi- 
nal vesicle  (corresponding  to  a nucleus),  with  its  small  excentric 
germinal  spot  (corresponding  to  a nucleolus). 

(/i,.)  Small  Unripe  Ova. — Besides  the  difference  in  size,  the 
follicles  show  no  separation  between  the  tunica  and  membrana 
granulosa.  They  form  a layer  of  cells  in  which  the  small  ovum 
appears  to  be  embedded. 

2.  The  Ovum  (fig.  361). — A fresh  ovum  may  be  obtained  thus. 
Take  the  ovary  of  a cow  or  sheep,  which  on  its  surface  shows  clear 

Cells  of  discus  proligerus. 

Germinal 
spot. 


Accessory 
nucleoli 
n.,  also/. 


Fig.  361.— Ripe  Ovum  of  Rabbit  highly  magnified. 

elevations  about  the  size  of  peas,  filled  with  a watery-looking  fluid ; 
these  are  ripe  follicles.  Prick  the  follicle  and  receive  its  contents 
upon  a slide.  The  liquor  folliculi  escapes,  and  with  it  the  ovum 
surrounded  by  the  cells  of  the  tunica  granulosa.  With  (L)  search 
for  the  ovum  in  the  fluid  on  the  slide.  Do  not  cover  the  prepara- 
tion while  doing  so.  If  a cover-glass  be  applied,  two  strips  of  paper 
must  be  placed  under  the  cover-glass  to  avoid  pressure  upon  the 
delicate  ovum.  Observe  the  ovum,  and  in  it  will  be  seen  the  parts 
already  described  and  shown  in  fig.  361. 

3.  T.S.  Fallopian  Tube  (L  and  H).  — This  is  hardened  in 
Muller’s  fluid  or  Flemming’s  fluid.  Treat  it  in  the  same  way  as 


THE  OVARY. 


XXXVI.] 


391 


the  intestine.  Make  transverse  sections,  and  stain  1 liem  either 
with  picro-carmine  or  logwood. 


Coimective 

tissue 

Ciliated 

epithelium. 

Circular  mus- 
cular fibres. 

Muscular  fibres, 
t;ut  across. 


Fig.  362. — T.S.  of  Fallopian  Tube. 

The  Fallopian  tubes  in  animals  often  pursue  a curved  course. 
The  peritoneum  should  he  cut  off 
as  close  to  the  tube  as  possible, 
and  the  latter  stretched  if  complete 
T.S.  are  required. 

(a.)  Observe,  most  external,  the 
thin  serous  coat. 

coat,  composed  of  non-striped 
muscle,  a very  thin  longitudinal 
layer  of  fibres,  and  a much  stronger 
circular  layer. 

(c.)  A thin  submucous  coat. 

(d)  The  mucous  coat,  thrown 
into  numerous  ridges  or  folds,  i.e., 
sections  of  longitudinal  folds,  so 
that  the  lumen  of  the  tube  appears 
somewhat  star-shaped  or  branched 
(fig.  362).  Sometimes  the  pro- 
cesses of  the  mucous  membrane 
are  very  complex  in  their  arrange- 
ment, and  give  rise  to  arborescent- 
looking  folds  in  transverse  sections  (fig.  363). 

(e.)  (H)  The  thick  mucous  coat  is  covered  by  a single  layer  of 


(b.)  Inside  this  the  muscular 


Fig.  363  —T.S.  of  the  Fimbriated  F.xtre- 
mity  of  the  Fallopian  Tube  of  Pig. 


392 


PRACTICAL  HISTOLOGY. 


[xxxvi. 


low  cylindrical  ciliated  cells,  but  there  are  no  glands,  although  the 
depressions  act  the  part  of  glands.  Under  this  is  connective  tissue 
with  a very  thin  muscularis  nmcosae. 

4.  T.S.  Fimbriated  End  of  Fallopian  Tube  stained  in  borax- 
carmine  and  cut  in  paraffin.  Observe  the  very  complex  folds  of 
the  mucous  membrane,  each  with  secondary  folds  and  covered  by 
ciliated  epithelium.  The  folds  are  much  higher  and  far  more  com- 
plex than  they  are  in  the  Fallopian  tube  (fig.  363). 

UTEKUS. 

The  wall  of  the  uterus  is  composed  of  smooth  muscle  lined  by 
a mucous  membrane.  As  a rule,  two  layers  of  muscular  fibre — 
imperfectly  separated  from  each  other — can  be  distinguished  in  the 
muscular  coat ; the  external  layer  having 
an  irregular  course ; then  follow  blood- 
vessels and  connective  tissue,  which  sepa- 
rate it  incompletely  from  the  inner  one 
arranged  more  circularly.  This  thick 
inner  layer  of  smooth  muscle  is  said  to 
represent  a much  hypertrophied  muscu- 
laris mucosae.  The  mucous  membrane 
(i  mm.  thick)  consists  of  connective  tissue 
containing  a very  large  number  of  cells 
and  nuclei,  and  embedded  in  it  are 
tubular  glands — utricular  glands — some 
of  them  simple,  and  others  branched  (fig. 
364),  especially  at  their  lower  ends. 
These  glands  seem  to  be  devoid  of  a 
membrana  propria,  but  the  surrounding 
connective  tissue  is  arranged  around  them 
in  a concentric  manner.  The  body  of  the 
uterus,  upper  half  of  the  cervix,  and  the 
uterine  glands  are  lined  by  a single  layer 
of  short  columnar  ciliated  epithelium. 
The  vaginal  portion  of  the  uterus  is  lined 
by  stratified  squamous  epithelium,  and 
carries  small  vascular  papillae  covered  in 
by  the  epithelium.  The  mucous  mem- 
brane rests  directly  on  the  muscular  coat, 
there  being  no  proper  submucosa.  Small 
processes  of  smooth  muscle  from  the 
muscular  coat  are  prolonged  inwards  between  the  bases  of  the 
glands.  The  mucous  coat  is  very  vascular  and  contains  many 
lymphatics. 


XXXVII.] 


MAMMARY  GLAND. 


393 


Methods. — It  is  rare  to  obtain  the  human  uterus  sufficiently 
fresh  for  microscopical  preparations.  Harden  the  uterus  of  a 
bitch,  cat,  or  rabbit,  in  chromic  and  spirit  fluid,  alcohol,  or  Miiller’s 
fluid.  Make  T.S.,  and  treat  them  as  the  Fallopian  tube. 

T.S.  Uterus. — (a.)  Observe  the  serous,  muscular,  and  mucous 
coats  as  in  the  tube,  but  here  the  muscular  coat  is  very  thick,  and 
is  composed  of  numerous  fibres  arranged  in  bundles  and  running  in 
all  directions.  The  arrangement  of  these  bundles  is  much  simpler 
in  animals  than  in  the  human  uterus. 

(b.)  The  mucosa  is  very  thick,  and  is  covered  by  a single  layer 
of  cylindrical  nucleated  cells,  and  has  numerous  glands,  which  are 
lined  by  similar  cells.  Hot  unfrequently  the  gland-tubes  branch, 
especially  near  their  lower  extremities.  It  is  difficult  to  retain  the 
cilia  on  the  epithelium  lining  the  cavity  of  the  uterus  and  its  glands. 
Between  the  tubules  is  a relatively  large  amount  of  connective 
tissue  containing  many  nucleated  corpuscles  and  blood-vessels.  As 
the  glands  pursue  a curved  course,  and  do  not  always  run  at  right 
angles  to  the  mucous  surface,  it  is  difficult  to  obtain  a section 
through  the  entire  length  of  a gland  (fig.  364). 


LESSOH  XXXVII. 

MAMMARY  GLAND,  UMBILICAL  CORD,  AND 
PLACENTA. 

The  mammary  gland  is  a compound  racemose  gland,  but  it  has 
about  twenty  galactoferous  ducts  which  open  on  the  nipple,  each 
duct  being  dilated  into  a small  reservoir  just  before  it  ends  on  the 
surface.  The  ducts,  when  traced  backwards,  branch  and  end  in 
acini  or  saccular  alveoli.  The  alveoli — as  in  all  glands — vary  in 
appearance  according  as  the  gland  is  or  is  not  active.  The  walls  of 
the  ducts  and  acini  consist  of  a basement  membrane,  said  to  be 
composed  of  branched  cells,  which  in  the  acini  is  lined  by  a single 
layer  of  somewhat  flattened  polyhedral  secretory  cells.  A cluster 
of  acini  gives  origin  to  one  of  the  larger  ducts,  and  a considerable 
amount  of  connective  tissue  lies  between  groups  of  acini.  In  fact, 
the  connective  tissue  greatly  preponderates.  During  lactation  the 
secretory  cells  are  taller  and  larger,  and  in  their  interior — probably 
formed  from  and  by  the  protoplasm  of  the  cells  themselves — are 
formed  the  fatty  granules  which  are  discharged  to  form  the  milk 
35 


394 


PKACTICAL  HISTOLOGY. 


[xxxvil. 


globules.  The  active  gland  presents  some  resemblance  to  a salivary 
gland,  so  closely  are  the  alveoli  pressed  together,  with  only  a small 
quantity  of  interstitial  connective  tissue  between  them.  The  acini 
are  in  groups  and  separated  from  each  other  by  fibrous  imperfect 

septa.  Numerous  cor- 
puscles, including  granu- 
lar cells,  occur  in  the 
alveolar  connective  tis- 
sue. The  ducts  are 
lined  by  columnar  epi- 
thelium, and  in  a section 
of  a human  gland  they 
appear  large. 

(i.)  Harden  small 
parts  of  the  mammary 
gland  in  absolute  al- 
cohol. Select,  when 
possible,  the  gland  of 
a recently  pregnant 
woman  (or  animal). 
Stain  the  sections  in 
hsematoxylin  and  mount 
in  balsam,  le.,  to  get  a general  view  of  the  gland  structure.  Stain 
in  bulk,  embed  and  cut  in  paraffin. 

(ii.)  Harden  very  small  pieces  of  a fresh  gland,  e,g.^  from  a 


Fia.  365.— T.S.  Mammary  Gland  D.  Duct ; A.  Group 
of  acini  with  much  connective  tissue  between,  x 20 


pregnant  cat  or  rabbit,  in  Flemming’s  mixture  and  stain  the 
sections  (very  thin)  in  safranin. 

1.  V.S.  Mammary  Gland  {Hcematoxylin)  (L  and  H)  (fig.  365). 
(a.)  Observe  the  groups  of  acini,  separated  by  a relatively  large 


XXXVII.]  UMBILICAL  CORD  AND  PLACENTA. 


395 


amount  of  somewhat  loose  connective  tissue,  and  sections  of  the 
ducts  (D).  The  sections  should  be  made  so  as  to  include  th.e 
nipple,  when  the  larger  ducts  with  their  dilations  will  be  seen. 
The  ducts  are  large  between  the  lobules,  and  within  the  latter  the 
course  of  the  finer  ducts  can  readily  be  traced. 

(6.)  The  globular  acini,  with  a basement  membrane  lined  by  a 
single  layer  of  somewhat  flattened  or  cubical  epithelium.  In  the 
inter-alveolar  tissue  many  leucocytes  and  granular  cells. 

2.  Active  Mammary  Gland  {Safranin)  (H). 

{a.)  Study  specially  the  acini.  Observe  the  large  and  tall 
columnar  cells  lining  the  acini,  and  in  some  of  the  cells  clear 
refractive  granules  of  fat.  The  lumen  is  wide,  and  is  usually 
partially  filled  with  the  debris  of  the  secretion-milk.  Osmic  acid  is 
a good  agent  for  showing  the  presence  of  fatty  granules  (fig.  366). 

3.  Colostrum  (H),  Le.,  the  first  milk  secreted  after  delivery. 
If  this  can  be  obtained,  examine  it,  and  note,  in  addition  to  the 
ordinary  milk-globules  (Lesson  I.  3),  large  coarsely  granular  nucle- 
ated retractile  cells — colostrum  corpuscles.  The  granules  are 
sometimes  pigmented,  and  are  fatty  (fig.  367). 

UMBILICAL  CORD  AND  PLACENTA. 

4.  T.S.  Umbilical  Cord. — Harden  this  in  Muller’s  fluid  or 
alcohol.  Make  T.S.  by  freezing,  and  stain  them  with  hsematoxylin 
or  picro-carmine.  Methyl-violet  is  also  a good  stain. 

(a.)  Note  on  the  outside  of 
the  circular  mass  of  tissue  a 
thin  layer  of  flattened  cells 
derived  from  the  amnion. 

{b.)  The  cord  itself,  composed 
of  Wharton’s  jeUy,  enclosing 
usually  two  umbilical  arteries 
and  a single  vein  with  very 
thick  muscular  coats.  They 
are  completely  surrounded  by 
Wharton’s  jelly,  which,  how- 
ever, in  a cord  at  full  time  is 
very  largely  composed  of  fibrous 
tissue.  Still  numerous  branched 
connective  tissue  corpuscles  exist 
in  the  meshes,  and  there  are 
also  present  numerous  lymphoid -looking  cells  (Lesson  XII.  10). 

5.  Fresh  Placenta  (H). — Tease  a fragment  of  a placenta  in 
normal  saline.  Note  the  vill%  each  long,  tapering,  and  branched. 
In  the  interior  capillary  loops  which  occupy  the  greater  part  of  the 


Fig.  368. — Human  Placenta  Villi.  Blood- 
vessels black. 


396 


TRACTICAL  HISTOLOGY. 


[XXXVIII 


villus,  so  that  only  a small  amount  of  connective  tissue  intervenes 
between  the  vessels.  Each  villus  is  covered  on  its  surface  by  a 
layer  of  epithelium,  which,  however,  is  thin  at  one  part  and  thick  at 
another.  Especially  at  the  ends  of  the  villi  are  large  granular  masses 
of  protoplasm  containing  many  nuclei,  but  one  cannot  make  out  a 
separation  of  these  masses  into  cells.  They  often  contain  vacuoles. 
The  arangement  of  the  blood-vessels  may  be  followed  from  the 
distribution  of  the  blood-corpuscles  (fig.  368). 

Small  portions  of  a placenta  are  also  to  be  hardened  in  Muller’s 
fluid  and  stained  in  bulk  in  borax-carmine.  Individual  villi  may 
be  isolated  in  dilute  alcohol  or  osmic  acid. 

6.  Injected  Placenta. — Examine  a vertical  section  of  a placenta 
with  the  foetal  blood-vessels  injected,  say  blue,  and  the  maternal 
vessels  red.  Observe  how  the  one  set  interlocks  with  the  other,  yet 
both  systems  are  closed  and  do  not  communicate  with  each  other. 


LESSOII  XXXYIII. 

TO  MAKE  PREPARATIONS  RAPIDLY  FROM 
FRESH  TISSUES. 

It  is  of  the  utmost  importance  that  the  student  should  be  acquainted 
with  the  methods  of  making  preparations  from  fresh  tissues  placed 
in  his  hands.  The  following  is  an  outline  of  the  work  that  each 
one  can  readily  do  for  himself  if  supplied  with  a pithed  frog,  or 
other  suitable  material. 


A.  From  a Frog. 

1.  Comeal  Corpuscles. — With  a sharp  pair  of  scissors  cut  out 
the  cornea.  Divide  it  into  two  parts. 

(a.)  Treat  one  by  the  lemon-juice  method  (p.  79). 

(/>.)  Treat  the  other  part  by  placing  it  direct  into  .5  per  cent. 
AuClg  (half  an  hour)  ; wash  in  distilled  water ; place  in 
a saturated  solution  of  tartaric  acid  at  50°  C.  until  the 
gold  becomes  reduced  (p.  79). 

(c.)  A cornea  may  be  placed  fresh  in  dilute  methylene-blue 
(i  : 300  normal  saline).  Mount  in  picrate  of  ammonia 
glycerine  (p.  192). 

2.  Comeal  Lymph-Spaces. — Remove  the  eyelids,  expose  the 
surface  of  the  other  cornea,  scrape  off  the  epithelium,  and  rub  it 


FRESH  TISSUES. 


XXXVIll.] 


397 


with  solid  silver  nitrate.  Cut  out  the  cornea  and  expose  it  to  light 
in  water  (p.  77). 

3.  Tendons. — These  are  best  made  from  the  tarsal  tendons, 
which  can  readily  he  snipped  off  in  considerable  lengths. 

(a.)  Fibrils. — Tease  a piece  in  baryta-water  and  mount  in 
glycerine. 

{h.)  Tendon  Cells. — (i.)  Add  dilute  acetic  acid  to  bring  into 
view  the  rows  of  cells,  then  wash  with  water,  and  after 
all  the  acid  is  removed  stain  with  logwood  or  picro- 
carmine.  (ii.)  Also  tease  a piece  in  normal  saline  con- 
taining a trace  of  methyl-violet. 

(c.)  Silver  one  of  the  tendons  to  show  the  endothelium  cover- 
ing its  surface  (p.  [66). 

(c/.)  Place  a fresh  tendon  in  ammoniacal  carmine  ( i o-  r 5 mins. ), 
wash  and  place  in  very  dilute  Delafield’s  logwood 
(10-15  mins.).  Wash,  tease;  and  mount  in  balsam. 
The  tendon  cells  are  red,  their  nuclei  blue,  and  the 
tendon  fibres  rosy. 

4.  Aponeurosis.— The  best  is  the  femoral. 

(a.)  Remove  the  membrane  and  stretch  it  on  a slide  by  the 
“semi-desiccation”  method  (p.  159),  and  after  it  is 
fixed  to  the  slide  apply  a drop  of  acid  methyl-green, 
or  normal  saline  with  methyl-violet.  The  nuclei  are 
thereby  stained,  and  the  crests  and  ridges  of  the  cells 
are  made  visible. 

(h.)  It  may  be  fixed  rapidly  on  a slide  with  absolute  alcohol  and 
stained  with  logwood.  Or  osmic  acid  may  be  used  to  fix  it. 

(c.)  Show  the  effect  of  acetic  acid. 

5.  Areolar  Tissue. — (a.)  Dissect  out  some  from  the  inter- 
muscular septa  of  the  leg  muscles.  Stain  with  methyl-violet  in 
normal  saline.  This  stains  the  cells.  Or  use  acetic-fuchsin  (p.  92). 

6.  Yellow  Elastic  Fibres.  — These  are  found  in  the  septa  between 
the  lymph-sacs.  Cut  .out  a septum,  fix  it  on  a slide  by  “ semi- 
desiccation,” and  then  add  acetic  acid.  Or  make  another  prepara- 
tion and  stain  it  with  a weak  solution  of  methyl-violet- 5 B (p.  93). 

7.  Pigment-Cells. — (a.)  These  are  found  in  the  web  of  the  frog’s 
foot.  Stretch  the  web  between  the  toes,  harden  it  in  absolute 
alcohol  for  an  hour  or  so,  peel  off  the  skin,  and  mount  it  in  balsam 

(p- 173)- 

{h.)  Or  use  the  mesentery,  or  almost  any  blood-vessel:  add 
dilute  acetic  acid  and  mount  in  glycerine. 

8.  Hyaline  Cartilage. — (a.)  Use  either  the  episternum,  scraping 
off  the  perichondrium,  or  make  a section  of  the  articular  cartilage 
on  the  femur  or  tibia.  Stain  in  hsematoxylin.  Or,  before  cutting, 
the  cartilage  may  be  hardened  for  an  hour  in  absolute  alcohol 


398 


PRACTICAL  HISTOLOGY. 


[XXXVIII. 


(b.)  A silver  nitrate  preparation  may  also  be  made  (p.  151). 

9.  Endothelium  of  Mesentery. — Place  pieces  of  mesentery  in 
AgXOg  (.25  per  cent.)  for  half  an  hour,  wash  in  distilled  water  and 
expose  to  light  in  50  per  cent,  alcohol 

Part  may  be  afterwards  stained  in  haematoxylin. 

10.  Endothelium  of  Great  Lymph-Sac. — Open  the  abdomen 
from  the  front  along  the  middle  line,  turn  aside  the  intestines, 
and  note  the  kidney.  A thin  membrane  or  septum  stretches  from 
this  to  the  abdominal  wall.  With  a fine  pipette  filled  with  silver 
nitrate  solution  perforate  this  membrane  and  allow  silver  nitrate  to 
flow  into  the  great  lymph-sac.  Expose  the  membrane  to  light,  and 
tlien  examine  in  glycerine  to  see  endothelium  and  stomata  (p.  239). 
One-half  may  be  stained  with  haematoxylin  to  show  the  nuclei.  Or 
expose  the  septum  from  behind  as  directed  at  p.  238. 

11.  Adipose  Tissue. — Use  the  yellow-coloured  fat  bodies  found 
in  the  abdominal  cavity. 

(a.)  Tease  a piece  in  glycerine. 

(b.)  Use  osmic  acid  (p.  169). 

See  also  other  methods  in  Lesson  XII. 

12.  Striped  Muscle. — In  this  one  must  demonstrate — 

(a.)  Sarcolemma  (p.  193). 

(b.)  Nuclei,  e.g.^  by  acetic  acid  (p.  194). 

(c.)  Sarcous  substance  with  its  cross  stripes.  Harden  for  half 
an  hour  in  alcohol  and  stain  with  haematoxylin  or  picro- 
carmine,  or  both.  Mount  in  glycerine.  Osmic  acid 
also  “ fixes”  the  striation. 

(d.)  Fibres  may  be  isolated  by  means  of  33  per  cent,  caustic 
potash,  but  they  must  be  examined  in  the  same  solu- 
tion. 

13.  Cardiac  Muscle. — Isolated  cells  are  obtained  by  the  33  per 
cent,  caustic  potash  method.  The  fresh  tissue  teased,  stains  well  in 
picro-carmine. 

14.  Smooth  Muscle. — Use 

(a.)  Frog’s  bladder  (p.  190).  In  addition,  spread  out  the 
bladder  on  a slide,  expose  it  to  the  vapour  of  glacial 
acetic  acid,  wash  away  the  epithelium,  stain  with 
violet-B,  and  mount  in  picrate-glycerine  (S,  Mayer), 

(b.)  Intestine.  The  muscular  coat  alone  is  to  be  used,  after 
scraping  away  the  mucous  coat.  Treat  it  as  above. 

15.  Epithelium. — Scrape  any  epithelial  surface,  diffuse  the 
scrapings  in  normal  saline  and  examine  fresh,  and  seal  up  with 
paraffin  wax. 

Squamous, — Use  cornea. 

Columnar, — Use  intestine. 

Ciliated, — Mucous  membrane  of  palate. 


FRESH  TISSUES. 


399 


XXXVIII.] 

Stain  others  with  acid  methyl-green  or  picro-carmine. 

INIake  cover-glass  preparations  (p.  140). 

Before  staining  pass  the  cover-glass  three  times  through  the  flame 
of  a Bunsen-burner. 

16.  MeduUated  Nerve-Fibres. — Expose  the  sciatic  nerve. 

(a.)  Tease  out  a few  fibres  and  show  them  fresh  in  normal 
saline.  Seal  up  the  preparation  with  paraffin  wax. 

(b.)  Tease  a piece  in  i per  cent,  osmic  acid,  cover  with  a watch- 
glass,  and  after  half  an  hour  mount  in  glycerine.  This 
blackens  the  myeline.  A part  of  this  may  be  stained 
in  picro-carmine  (p.  206). 

(c.)  Tease  a piece  in  .5  per  cent,  silver  nitrate  for  Eanvier’s 
crosses  (p.  207). 

(d.)  Harden  a piece  of  nerve  in  alcohol  for  a quarter  of  an 
hour.  Tease,  place  in  ether  for  10  minutes,  transfer  to 
alcohol.  Stain  wdth  logwood  and  mount  in  glycerine. 
This  shows  the  axis-cylinder  and  nuclei  of  sheath. 

(e.)  Place  a piece  of  fresh  nerve  in  collodion  to  show  axis- 
cylinder.  This  preparation  only  lasts  for  a short  time 
(p.  211).  _ 

(/.)  Show  with  silver  nitrate  the  endothelial  sheath  on  one  of 
the  small  nerves  to  be  found  in  the  dorsal  lymph-sac, 
stretching  between  the  back  muscles  and  the  skin 
(p.  207). 

17.  Peripheral  Nerve-Cells. — Use  the  rapid  gold  chloride 
(p.  79)  or  methylene-blue  method  (p.  222). 

(a.)  Cells  along  course  of  aorta.  Cut  out  the  abdominal  part 
of  the  aorta. 

(5.)  Cells  of  sympathetic  (p.  216)  or 

(c.)  Cells  of  spinal  ganglia,  or  other  ganglia  (p.  215). 

(d.)  Interauricular  septum  of  the  heart.  The  heart  must  be 
distended  and  kept  fixed  in  this  position  (p.  233). 

18.  Retina.  — Carefully  dissect  out  the  eyeball,  remove  retina, 
(a.)  Place  a part  in  i per  cent,  osmic  acid  and  then  make 

teased  preparations.  One  should  show  the  pigmented 
epithelium,  rods  with  outer  segments  blackened,  pieces 
of  the  several  layers,  the  glistening  fatty  globule 
rendered  brownish. 

(h,)  Harden  a piece  in  absolute  alcohol  and  tease  in  very 
dilute  eosin.  Mount  in  glycerine. 

(c.)  Place  small  pieces  in  very  dilute  methylene-blue  (p.  222) 
to  show  the  nervous  elements^  Mount  it  in  picro- 
glycerine  (p.  222). 

19.  Blood-Vessels. — A new  frog  will  be  required.  Proceed  as 
directed  at  p.  230. 


400 


PRACTICAL  HISTOLOGY. 


[XXXVIIL 


(a,)  Inject  AgNOg  (.5  per  cent.),  and  isolate  arteries,  veins, 
and  capillaries;  best  from  the  intestine.  Mount  in 
balsam. 

(h.)  Show  the  effect  of  acetic  acid  on  one  of  the  larger  vessels 
of  the  mesentery. 

(c.)  Methylene-blue  injected  into  the  vessels  shows  the  lining 
endothelium  and  nerve-fibres. 

20.  Blood  and  Blood-Corpuscles. 

{a.)  Osmic  acid  and  picro-carmine  (p.  110). 

(Z>.)  Cover-glass  preparations  stained  by  eosin  in  glycerine  and 
then  in  logwood.  Or  other  dyes  or  double  stain 
(p.  1 14). 

(c.)  Cover-glass  preparations  stained  by  methylene-blue  for  the 
nuclei.  Pass  the  cover-glass  three  times  through  the 
flame  of  a Bunsen-burner  before  staining. 

{d.)  The  colourless  corpuscles  may  be  stained  in  the  methylene- 
blue  specimens,  but  they  may  be  specially  stained  by 
eosin-glycerine  or  indulin-glycerine,  which  stain  certain 
granules  in  their  protoplasm  (p.  402). 

21.  Fibrin  (p.  1 1 9). 

22.  Marrow. — Squeeze  out  same. 

(a.)  Examine  fresh  in  NaCl  (.6  per  cent.)  with  methyl-violet. 

(1).)  Cover-glass  preparations  stained  with  eosin-glycerine  and 
logwood  (p.  188).  The  easiest  way  is  to  pass  the  cover- 
glass  with  its  adherent  film  of  marrow  three  times 
through  the  flame  of  a Bunsen-burner  before  staining. 

23.  Motor  Nerves  to  Muscles. — Gold  method  or  methylene- 
blue  (p.  219). 

B.  From  a Mammal. 

If  a mammal,  e.^.,  rat  or  guinea-pig,  or  part  thereof,  be  given 
from  which  to  prepare  specimens,  the  methods  are  much  the  same 
as  those  described  above. 

24.  Areolar  Tissue. — (a.)  To  show  its  histological  elements  the 
best  plan  is  to  inject  under  the  skin,  by  means  of  a hypodermic 
syringe,  some  fluid  which  will  form  an  artificial  oedema,  e.^.,  methyl- 
violet  in  normal  saline,  osmic  acid  (i  per  cent.),  silver  nitrate 
I : 300.  The  first  of  these  fluids  does  not  alter  the  tissues.  Excise 
a piece  and  examine  it  in  the*same  fluid. 

(h.)  Use  the  “ semi-desiccation  method  with  a small  piece  snipped 
off  and  spread  out  on  a slide. 

(c.)  Cell-spaces  by  means  of  AgNOg  (p.  162). 

In  addition  to  the  methods  described  at  p.  16 1 and  p,  162,  use 
acetic  acid  to  show  elastic  fibres ; magenta  to  stain  the  latter. 

25.  Tendons,  e.g.,  rat  (p.  168),  and  methods  at  p.  397. 


XXXVIII.]  FRESH  TISSUES.  4OI 

26.  Diaphragm. — (a.)  Show  its  endothelial  covering  by  means 
of  AgNO^,  and  (b)  its  lym]>hatics  (p.  237). 

27.  Adipose  Tissue. — One  must  demonstrate  the  cells  (Lesson 
XIL). 

(a.)  Fresh,  unaltered  in  normal  saline,  using  either  part  of 
omentum  or  fat-cells  from  under  skin.  Seal  up  the 
preparation  with  paraffin  wax. 

(h.)  Action  of  osmic  acid. 

(c.)  Action  of  alcohol  and  ether  to  remove  fats,  and  show 
empty  envelopes. 

(d.)  Harden  fat-cells  in  alcohol  and  stain  in  logwood  to  show 
n aclei. 

(e.)  Subcutaneous  injection  of  silver  nitrate  (i  : 500)  to  show 
general  characters  of  cells. 

28.  Granular  Cells  (“  Mastzellen ’’). — They  occur  in  large 
numbers  in  the  omentum  of  the  rat.  Place  a small  part  of  the 
omentum  in  a watch-glass  containing  aniline-water  and  20-30  drops 
of  a concentrated  alcoholic  solution  of  dahlia  or  gentian.  Heat  for 
short  time  as  directed  in  Lesson  X.  14.  Wash  in  distilled  water 
and  then  in  acid-alcohol  until  nearly  everything  is  decolorised 
except  the  granular  cells.  The  tissue  may  also  be  stained  with 
lithium-carmine.  Mount  in  balsam.  The  nuclei  of  the  cells  are 
red,  and  only  the  granules  in  the  protoplasm  of  the  granular  cells 
are  blue. 

The  mucous  membrane  of  a dog’s  tongue  hardened  in  alcohol  and 
treated  in  the  same  way  shows  numerous  granular  cells. 

29.  Red  Marrow  of  Bone. — Methods  (p.  188).  Use  the  ribs 

and  heads  of  long  bones  of  guinea-pig.  / 

The  dry  cover-glass  method  is  excellent.  Dry  them  in  flame  of 
spirit-lamp,  or  pass  them  three  times  through  the  flame  of  a Bunsen- 
burner.  Stain  with  eosin-glycerine  and  then  with  methylene-blue 
or  logwood.  Many  of  the  smaller  cells  will  show  eosinophile 
granules. 

For  studying  the  formation  of  the  elements  of  the  blood  in  red 
marrow  or  other  situations,  the  following  method  of  Foa  is  good.^ 
The  red  marrow  or  blood  is  “fixed”  in  the  following  fluid  after  it 
cools: — 100  cc.  Muller’s  fluid  is  heated  with  2 grams  of  mercuric 
chloride.  Keep  in  a thermostat  (2-3  hours)  at  35°  C.  Harden  in 
alcohol,  cut  sections,  and  stain  (1-3  minutes)  with  the  following  : — 


Haematoxylin  solution  (Bohmers)  , . . 25  cc. 

Safranin  i 2)er  cent,  watery-alcoholic  solution  . 20  ,, 

Water 100  ,, 


Then  stain  in  weak  picric  acid.  Xylol-balsam. 


2 G 


^ Zeits.  f,  wiss.  M Her  oak.  ^ ix.,  1892,  p.  227. 


402  PRACTICAL  HISTOLOGY.  [XXXVIII. 

30.  Blood  Crystals  (p.  120). — If  it  be  a guinea-pig  or  rat  use 
the  defibrinated  blood  to  obtain  blood  crystals. 

(a.)  Add  water  = haemoglobin  crystals. 

(b. ) Add  a small  quantity  of  ether  = haemoglobin  crystals. 

(c.)  Add  amyl  nitrite  = me thaemoglobin  crystals. 

31.  Blood— Ehrlich’s  Granules. — Perhaps  it  might  be  well  to 
give  here  a short  resume  of  some  of  the  results  of  Ehrlich  and  his 
pupils.  1 Cover-glass  preparations  of  the  blood  of  different  animals 
are  made,  and  they  are  either  exposed  to  the  air  to  dry  (or  they 
may  be  carefully  heated  for  several  hours  at  120°  C.  or  passed 
several  times  through  the  flame  of  a Bunsen-burner).  On  being 
dried  rapidly  in  the  air,  there  is  no  coagulation  of  the  cell-proteids, 
and  thus  the  cells  retain  their  natural  tendency  to  stain  with  dyes. 
As  haemoglobin  is  soluble  in  water  it  is  better  to  use  the  dyes  dis- 
solved in  glycerine. 

Leucocytes. — The  ‘‘granules”  present  in  the  protoplasm  in  the 
varieties  of  white  blood-corpuscles  vary  in  their  reaction  to  staining 
reagents.  Thus  some  are  stained  by  what  Ehrlich  calls  acidophile 
dyes,  of  which  eosin  is  one.  It  is  not  enough  that  the  granules  are 
stained  by  one  of  these  dyes.  As  a general  rule,  granules  which  are 
stained  by  all  the  following  solutions  belong  to  his  a-granulation 
class  and  are  “ eosinophilous  granules.” 

(1.)  Eosin  in  glycerine. 

(2.^  Glycerine  saturated  with  indulin. 

(3.)  Concentrated  watery  solution  of  orange. 

The  eosinophile  cells  (a  granules)  are  always  present  in  the  leuco- 
cytes of  frog’s  blood,  marrow  of  frog  (numerous) — very  few  in 
spleen — numerous  also  in  the  mesentery.  In  the  rabbit  they  occur 
in  the  blood,  marrow  (very  numerous),  spleen  (few). 

Make  a cover-glass  preparation  and  dry  it  either  at  120°  C. 
(several  hours)  or  rapidly  in  the  flame  of  a Bunsen.  Stain  for  an 
hour  (or  longer)  with  eosin-glycerine,  wash  in  water,  dry  and  mount 
in  balsam.  Or  stain  cover-glass  preparations  in  glycerine  (30  cc.) 
containing  2 grams  each  of  aurantia,  indulin,  and  eosin.  Or  a 
saturated  alcoholic  solution  of  bluish-eosin  may  be  used. 

If  an  eosin-indulin  glycerine  solution  be  used,  the  a-granulations 
are  purplish-red  and  the  nuclei  well  stained  bluish-black  by  the 
indulin. 

The  granular  cells  (‘‘  Mastzellen  ”),  which  occur  so  abundantly 
“in  the  connective  tissue  of  the  frog  and  some  other  animals,  also 
occut  in  the  blood  of  the  frog,  triton,  and  tortoise.  In  man,  accord- 
ing to  Ehrlich,  they  are  found  only  pathologically.  The  granules 

^ Farhenanalyt.  Unters,  z.  Histol.  u.  Klinik  des  Blutes^  by  P.  Ehrlich, 
Berlin,  1891. 


xxxviil] 


FRESH  TISSUES. 


403 


in  these  cells  are  stained  by  a fluid  composed  of  100  cc.  water,  50 
cc.  absolute  alcohol  saturated  with  dahlia,  10- 12. 5 cc.  glacial  acetic 
acid.  The  leucocytes  are  stained  blue,  while  the  granules  have 
what  Ehrlich  calls  a metachromic  red-violet  tint.  They  correspond 
to  Ehrlich’s  y-granulations,  and  have  been  specially  investigated  by 
Westphal  (lo(%  cit.^  p.  17). 

The  8-granulations  occur  especially  in  the  mononuclear  leucocytes 
of  human  blood.  They  are  stained  by  basic  dyes. 

The  e-granulations,  or  neutrophile  granules,  occur  in  the  poly- 
nucleated  elements  of  human  blood.  They  are  stained  only  by 
neutral  dyes,  e.y.,  acid-fuchsin  and  methyl-blue.  Ehrlich  classifies 
dyes  as  acidopliile,  e.y.,  eosin,  aurantia,  and  indulin ; neutrophile, 
e.g.,  acid-fuchsin  or  fuchsin-S,  methyl-blue ; hasophile,  e.g.,  dahlia, 
gentian-violet,  fuchsin. 

Ehrlich  also  calls  granules  which  attract  acid  dyes  “ oxyphile  ” — 
a term  adopted  by  Wright  and  Bruce,  ^ whose  method  is  described 
below.  According  to  the  latter  observers,  the  nucleus  of  the  leuco- 
cyte is  invariably  basophile,  while  the  granules  of  normal  leucocytes 
are  oxyphile. 

Staining  of  Oxyphilous  or  Eosinophilous  Granules. — Cover- 
glass  preparations  are  fixed  either  by  dry  heat  (Ehrlich’s  method) 
or  by  chemical  reagents  (osmic  acid,  HgCl2). 

Float  the  cover-glass  on  a i per  cent,  watery  solution  of  eosin 
(^-i  min.).  If  it  be  desired  to  stain  even  more  rapidly,  add  a trace 
of  acetic  acid  to  the  fluid,  when  the  preparation  rapidly  becomes 
over-stained.  The  surplus  dye  can  be  removed  from  all  parts  of  the 
cells,  except  the  oxyphile  or  eosinophilous  granules,  by  dipping  the 
cover-glass  into  a very  dilute  solution  of  sodic  carbonate. 

Basophilous  Granules. — These  are  best  stained  with  Loeffler’s 
methylene-blue,  which  stains  all  basophilous  elements,  e.g.,  nuclei 
and  basophile  granules.  It  may  dissolve  out  oxyphilous  granules. 
If  the  specimen  has  been  already  stained  with  eosin  (the  excess 
extracted  by  weak  alkali),  then  only  a second  or  so  is  required  to 
stain  with  methylene-blue.  Thus  with  care  it  is  possible  to  stain 
the  oxyphile  and  basophile  elements  of  the  leucocyte. 

These  observers  deny  the  existence  of  so-called  neutrophile 
granules.  They  believe  them  to  be  really  oxyphilous  in  their 
behaviour  to  stains. 

32.  Stained  Leucocytes. — Either  one’s  own  blood  or  the  blood  of 
an  animal,  or  the  leucocytes  of  lymph-glands,  may  be  used.  Use  the 
dry  cover-glass  method,  passing  the  cover-glass  three  times  through 
the  flame  of  a Bunsen-burner  before  staining.  Excellent  prepara- 
tions of  the  nuclei  stained  blue  are  obtained  by  methylene-blue 


^ BriU  Med,  Jour.,  Feb.  1893. 


404 


PRACTICAL  HISTOLOGY. 


[XXXVIIL 


alone,  or  first  stain  in  eosin  and  then  in  methylene-blue  or 
hsematoxylin.  For  leucocytes  the  blood  of  the  horse  is  specially 
valuable,  as  the  white  cells  are  so  large  (^Sherrington). 

33.  Fibrin,  Haemin,  Cartilage,  Muscle,  Nerve,  and  the  other 
tissues  are  treated  as  recommended  for  frog^s  tissues,  and  the  same 
is  the  case  with  organs. 

34.  Salivary  Glands  and  Pancreas. 

Tease  a small  part  of  the  parotid,  or  other  salivary  gland,  or 
the  pancreas,  in  aqueous  humour  to  see  the  fresh  condition  of  these 
glands.  In  the  guinea-pig’s  glands  note  the  zymogen  granules. 

A permanent  preparation  may  be  made  by  exposing  small  pieces 
to  the  vapour  of  osmic  acid  and  mounting  in  glycerine.  In  this 
way  the  zymogen  granules  are  preserved  (Lesson  XXIII.  p.  265). 

N.B. — In  this  Lesson  the  methods  stated  expressly  exclude  com- 
plicated methods  of  hardening  and  section-cutting. 


ADDENDA. 

A.  Altmann’s  Researches  on  ‘‘  Granula  ” in  CeUs. 

For  those  who  wish  to  study  Altmann’s  views  on  the  constitution 
of  the  protoplasm  and  nuclei  of  cells,  and  the  methods  of  displaying 
what  he  calls  his  “granula,”  we  must  refer  to  his  monograph, 
entitled  Die  Elemerdarorganismen  und  ihre  Beziehung  zu  den  Zellen^ 
Leipsig,  1890,  which  contains  twenty-one  beautiful  plates. 

B.  Obreggia’s  Method  for  Paraffin  Sections. 

The  method  of  Obreggia  Centrdlhlatty  1890)  has 

been  applied  by  Gulland  (^Journal  of  Pathology ^ 1893)  to  paraffin 
sections.  The  hardened  tissues  are  embedded  in  paraffin,  and 
ribbons  of  sections  are  cut  with  a rocking  microtome  or  Minot’s 
form.  The  sections  are  at  once  transferred  to  glass  plates  coated 
with  the  following  solution  : — 

Syrupy  solution  of  powdered  candy-sugar  made  with  boiling 

distilled  water  . . . . . . . . 30  cc. 

Absolute  alcohol  . . . . . . . . 20  ,, 

Transparent  syrupy  solution  of  pure  dextrin  made  with  dis- 
tilled water 10,, 

Pour  this  solution  over  the  plates  two  or  three  days  before  they  are 


ADDENDA.  405 

used ; run  off  the  excess ; allow  the  plates  to  dry  slowly  in  a hori- 
zontal position  and  protected  from  dust. 

Arrange  the  series  of  sections  in  rows  on  the  plates.  Place  the 
plates  in  a paraffin-oven,  which  is  kept  at  a temperature  slightly 
above  the  melting-point  of  the  paraffin  employed,  and  leave  it  there 
for  a few  minutes,  when  the  embedded  tissues  stick  fast  to  the 
prepared  surface. 

Remove  the  surplus  paraffin  with  xylol  or  naphtha,  and  then 
wash  with  methylated  spirit  or  absolute  alcohol. 

The  spirit  is  run  off,  and  the  plates  are  covered  with  the  following 
celloidin  solution : — 

Phytoxylin 6 grm. 

Absolute  alcohol  . . . . . 100  cc. 

Pure  eiher >> 

The  plates  are  placed  horizontally.  After  the  thin  sheet  of  celloidin 
solidifies,  run  a knife  along  between  the  rows  of  sections,  and  allow 
further  evaporation  to  take  place. 

When  the  sections  are  required  plunge  the  plate  into  water ; the 
ribbons  float  off  as  the  sugar  is  dissolved.  The  ribbons  may  be 
stained  with  any  reagents  except  those  which  dissolve  or  overstain 
celloidin.  Stain  with  very  dilute  Ehrlich^s  acid-haematoxylin  (p. 
69),  and  then  wash  in  dilute  eosin.  Dehydrate  the  sections,  and 
clarify  in  a mixture  of  xylol  3 parts  and  carbolic  acid  crystals  i part, 
and  mount  in  balsam.  We  have  tried  this  method,  as  recom- 
mended by  Gulland,  and  find  that  it  works  very  well.  Moreover, 
we  found  that  the  sections,  after  being  .floated  off,  can  be  kept  in 
80  per  cent,  alcohol  until  they  are  required. 


APPEI^DIX. 


A.— SOME  WORKS  OF  REFERENCE. 

A. — Systematic  Histology. 

Schwann,  Mikrosk.  Untersuch.,  1838  (translated  by  the  Sydenham  Society 
1847). — R.  Virchow,  Die  Cellular  Pathologie  (translated  by  Chance)  i860. — 
Henle,  Handbuch  der  systematischen  Anatomie  des  Menschen,  3rd  ed., 
1866-83. — W.  Krause,  Allgemeine  und  mikroskopische  Anatomie,  Han- 
nover, 1876. — F.  Leydig,  Lehrbuch  der  Histologie  des  Menschen  und 
der  Thiere,  Hamm,  1857  ; his  Untersuchungen,  1883 ; and  his  Zellen. 
Gewebe,  Bonn,  1885. — L.  Ranvier,  Traite  technique  d’histologie,  Paris,  2nd 
ed.,  1889. — G.  Schwalbe,  Lehrbuch  der  Neurologie,  Erlangen,  1881  ; Lehrb. 
d.  Anat.  d.  Sinnesorgane. — S.  Strieker,  Handbook  of  Histology  (trans- 
lated by  the  Hew  Sydenham  Society),  1871-73. — L.  Beale,  The  Struc- 
ture of  the  Elementary  Tissues,  London,  1881. — A.  Kdlliker,  A Manual  of 
Human  Microscopic  Anatomy,  London,  i860 ; and  his  leones  Histolog., 
Leip.,  1864;  vol.  i.  of  his  Handbuch  d.  Gewebelehre,  Leipzig,  1889. — 
Rindfleisch,  A Manual  of  Pathological  Anatomy  (translated  by  R.  Baxter), 
London,  1873.— C.  Toldt,  Lehrbuch  der  Gewebelehre,  Stuttgart,  3rded.,  1888. 
— E.  Klein  and  E.  Noble-Smith,  Atlas  of  Histology,  London,  1872. — 
H.  Prey,  Handbuch  der  Histologie  und  Histochemie  des  Menschen,  Leipzig, 
1876,  Grundziige,  1885. — Cadiat,  Traite  d’anat.  gen.,  Paris,  1879. — Brass, 
Kurzes. Lehrbuch  d.  Histologie,  Leipzig,  1888. — Heitzmann,  Mikrosk.  Mor- 
phology, 1882. — Purser,  Man.  of  Hist.,  Dublin. — E.  Klein,  Elements  of 
Hist.,  London,  1883. — W.  Flemming,  Zellsubst.  u.  Zelltheilg,  Leipzig,  1882. — 
Bizzozero,  Hand.  d.  klin.  Mikroskop.,  Erlang.,  2nd  ed.,  1888. — Carnoy,  Gilson, 
and  Denys,  Biol.  Cellul.,  Louvain,  1884-88. — Renaut,  Traite  d’Histologie,  Paris, 
1885. — Frommann,Unters.  11.  thier.  u.  pflanz.  Zellen, Jena,  1884. — Wiedersheim, 
Lehrb.  d.  vergl.  Anat.,  Jena,  1888. — S.  L.  Schenk,  Grundriss  der  Histologie 
d.  Menschen,  Vienna,  1885. — Orth,  Cursus  d.  norm.  Histol.,  4th  ed.,  1886. — 
S.  Mayer,  Histolog.  Taschenbuch,  Prag.,  1887. — Stbhr,  Lehrb.  d.  Histol.,  5th 
ed.,  Jena,  1892.— Lee  and  Henneguy,  Traite  de  meth.  d’Anat.,  Paris,  1888. — 
Schafer,  Essentials  of  Histology,  3rd  ed.,  1892.  — Owsjannikow,  Text-Book 
of  Histology  (Russian),  1888. — Fusari  and  Monti,  Compendio  di  Istologia 


APPENDIX. 


407 


generale,  Torino,  1891. — Ellenberger,  Vergleich.  Histol.  der  Hausthiere, 
voL  i.,  Berlin,  1887,  vol.  ii.,  1892. — Altmann,  Die  Elementarorganismen  u. 
ihre  Beziehung  z.  d.  Zellen,  Leipzig,  1890  (with  21  plates).  An  abstract  of 
the  methods  by  which  Altmann  prepares  his  “ granula  ” in  cells  will  be  found 
in  Zeit.  f.  mik.  Anat.,  vii.  p.  199,  1890. — Quain’s  Anatomy,  loth  ed.,  edited 
by  Schafer  and  Thane,  1892-93. — Obersteiner,  Anleitung  b.  Studium  d. 
Baues  d.  nerv.  Centralorg.,  2nd  Germ,  ed.,  1892.  English  ed.  translated  by 
Hill  from  1st  Germ.  ed. — Edinger,  Zwolf  Yorlesung.  v.  d.  Bau  d.  Central 
Nerv.  System,  2nd  ed.,  and  English  trans.  by  Vittum  and  Riggs,  1890. 

B. — The  Microscope,  Microscopical  Technique,  and  Manuals  of  Practical 

Histology. 

Some  of  the  works  already  mentioned  contain  descriptions  of  microscopical 
methods,  e.g.,  those  of  Ranvier,  Stohr,  Orth. 

Dippel,  Das  Mikroskop  und  seine  Anwendung. — Beale,  How  to  Work 
with  the  Microscope,  London,  1880. — H.  Frey,  Das  Mikroskop.,  8th  ed.,  1886. — 
Carpenter’s  The  Microscope  and  its  Revelations,  edited  by  Ballinger,  7th  ed., 
1892. — J.  Hogg,  The  Microscope,  12th  ed.,  1887. — Naegeli  and  Schwen- 
dener.  The  Microscope  in  Theory  and  Practice  (translated  from  2nd  Germ, 
ed.),  1892. — Gage,  The  Microscope  and  Microscopical  Methods,  Philadelphia, 
1892. — Amstrom,  Anleit.  z.  Benutz.  d.  Polaris- Mikroskop.,  Leip.,  1892. — 
Rutherford,  Outlines  of  Histology,  1876. — Schafer,  Pract.  Histol.,  London, 
1877. — W.  Stirling,  Text-Book  of  Pract.  Histol.,  London,  1881. — Foster  and 
Langley,  Pract.  Phys.,  5th  ed.,  1884. — Friedlander  and  Martinotti,  Tec- 
nica  microscopica,  Turin,  1885. — Garbini,  Manuale  per  la  tecnica  mod.  del 
Mikroscopio,  3rd  ed.,  1891. — Fol,  Lehr.  d.  vergleich.  mikros.  Anatomic,  Leip., 
Pt.  i.,  1885. — Behrens,  Tabellen  z.^Gebrauch  b.  mik.  Arbeiten,  Braunschweig, 
2nd  ed.,  1892. — Feamley,  Pract.  Histol.,  1887. — W.  Stirling,  Histological 
Memoranda,  Aberdeen,  1880. — Lee  and  Henneguy,  Traite  de  meth.  de  PAnat., 
Paris,  1888. — Friedlander  and  Eberth,  Mik.  Technik.,  4th ed.,  Berlin,  1888. 
— Gierke,  Farberei  z.  mik.  Zwecken,  Braun.,  1887. — Renaut,  Traite  d’Histol. 
pratique,  Paris,  1889. — Behrens,  Kossel,  and  Schiefferdecker,  Das  Mikro- 
skop., Pt.  L,  Braunschweig,  1889,  Pt.  ii.,  1891. — Ramon  y Cayal,  Manual  de 
Histologia  normal,  Valencia,  1889. — Bbhm  and  Oppel,  Taschenb,  d.  mik. 
Technik.,  Miinchen,  1890. — B.  Rawitz,  Leitfaden  f.  hist.  Untersuch.,  Jena, 
1889. — Neelsen,  Grundriss  d.  Path. -Anat.  Technik,  Stuttgart,  1892. — Squire, 
Methods  and  Formulae  used  in  the  Preparation  of  Animal  and  Vegetable 
Tissues  for  Micros.  Exam.,  London,  1892. — Strassburger,  Das  botan,  Practi- 
cum,  and  English  trans. — Zimmermann,  Die  botan.  Mikrotechnik,  Tubingen, 
1892. 

C. — Journals. 

Archiv  f.  mik.  Anatomic,  Bonn,  formerly  edited  byM.  Schultze  and  now 
by  Hertwig,  La  Valette,  St  George,  and  Waldeyer. — Archiv  fiir  Anat.  u. 


4o8 


APPENDIX. 


Physiol.,  edited  by  Du  Bois-Reymond. — Virchow’s  Archiv.— Quarterly  Micro- 
scopicalJournal,  London. — Journal  of  the  Royal  Microscopical  Soc.,  London. — 
Jour,  of  Anat.  and  Physiol.,  edited  by  Humphry,  Turner,  and  M‘Kendrick. — 
La  Cellule,  Louvain. — Journal  de  Micrographie,  Paris,  edited  by  Pelletan. — 
Zeits.  f.  wiss.  Mikrosk.,  edited  by  Behrens. — Zoolog.  Anzeiger,  edited  by 
V.  Cams. — Internats.  Monats.  f.  Anat.  u.  Phys.,  edited  by  Krause. — Journal 
of  Physiology,  edited  by  Foster. — Archiv  ital.  de  Biologic,  edited  by  Mosso. — 
Proceedings  and  Transactions  of  the  Royal  Society. — Comptes  rendus  de  PAcad. 
des  Sciences. — Sitzb.  d.  k.  Akad.  d.  Wissenschaft,  Wien. — Archiv  f.  d. 
gesanimte  Physiologic,  edited  by  Pfluger. — Journal  of  Morphology,  edited 
by  Whitman,  in  which  will  be  found  elaborate  papers  by  Minot  on  the  Uterus 
and  Placenta,  and  on  the  Ear  by  H.  Ayers. 


B.— TABLES  OF  MAGXIFYTIS^G  POWER  OF  OBJECTIYES 
AND  OCULARS. 


Objective. 

Magnifying 
Power  of  Ob- 
jective alone. 

Eyepiece  No.  i (A) 
magnifies  5 
times ; combined 
with  Objective 
magnifies 

Eyepiece  No.  2(B) 
magnifies  73 
times;  combined 
with  Objective 
magnifies 

Eyepiece  No.  4(D) 
magnifies  20 
times ; combined 
with  Objective 
magnifies 

1 inch 

10 

50 

75 

200 

A >> 

25 

125 

187 

500 

1 

T >> 

40 

200 

275 

800 

1 

6-  5> 

50 

250 

300 

1000 

i )> 

60 

300 

450 

1200 

t ’■ 

80 

400 

600 

1600 

tV  n 

100 

500 

750 

2000 

A 

120 

6co 

900 

2400 

This  table  is  calculated  for  a lo-inch  tube,  and  gives  approximately  the 
magnifying  power ; but  if  accuracy  be  required,  each  combination  of  lenses 
and  objectives  must  be  measured  by  the  method  already  described  at  page  19. 
(After  Gibhes.) 

Hartnack’s  Dry  Lenses. 


Objective. 

Ocular, 

No.  1. 

No.  2. 

No.  3. 

No.  4. 

4 

40 

50 

65 

100 

7 

150 

220 

300 

450 

8 

250 

360 

400 

600 

9 

360 

430 

520 

850 

APPENDIX. 


409 


Magnifying  Power  of  Zeiss’s  Objectives  and  Oculars. 


Objective. 

Length  of  Tube  155  mm. 
Ocular. 

No.  1. 

No.  2. 

No.  3. 

No.  4. 

A 

7 

II 

15 

22 

A* 

4-12 

7-17 

ia-24 

A,  AA 

38 

52 

71 

97 

B,  BB 

70 

95 

130 

175 

C,  CG 

120 

145 

195 

270 

D,  DD 

175 

230 

320 

435 

E 

270 

355 

490 

670 

F 

405 

540 

745 

1010 

The  lens  A*  is  a particularly  useful  low-power  lens,  as  by  merely  rotating 
a collar,  a great  variety  of  magnifying  power  is  obtained  without  changing 
the  lens. 


Magnifying  Power  of  Leitz’s  Objectives  and  Oculars. 


Number 

of 

Objective. 

Magnifying 
Power  of 
Objective 
without 
Ocular. 

Length  of  Tube  160  mm. 
Ocular. 

0 = 5.6. 

I.  =6.9. 

II.  =8.5. 

III.  = 12.7. 

IV.  = 16.3. 

V.  = 19. 1. 

I 

3-2 

16 

21 

25 

39 

50 

60 

2 

5 

26 

34 

40 

63 

81 

95 

3 

9 

47 

62 

72 

114 

146 

171 

4 

II 

58 

75 

88 

139 

179 

210 

5 

26 

137 

179 

208 

330 

423 

496 

6 

34 

180 

234 

272 

431 

554 

649 

7 

47 

250 

325 

380 

600 

770 

900 

8 

60 

3*8 

414 

480 

762 

978 

1146 

C.— LIST  OF  MAKEES  OF  MICEOSCOPES,  &c. 

Microscopes. 

American  Mahers  and  Agents. 

Bausch  & Lomb  Optical  Co. , Rochester,  N.  Y. 

J.  W.  Queen  & Co.,  1010  Chestnut  Street,  Philadelphia. 

Williams,  Brown  & Earle,  N.  E.  cor.  Tenth  and  Chestnut  Sts.,  Philadelphia. 
Eimer  & Amend,  205-21 1 Third  Avenue,  New  York. 

Joseph  Zentmayer,  209  S.  Eleventh  Street,  Philadelphia. 

36 


410 


APPENDIX. 


Foreign  Makers. 

Powell  & Lealand,  170  Euston  Road,  N.  W.,  London. 
R.  & J.  Beck,  68  Cornhill,  E.  C.,  London. 

Swift  & Son,  81  Tottenham  Court  Road,  London. 

C.  Baker,  244  High  Holborn,  London. 

H.  Crouch,  66  Barbican,  London. 

Carl  Zeiss,  Jena. 

E.  Hartnack,  Waisenstrasse,  39,  Potsdam. 

W.  & H.  Seibert  (successors  to  Gundlach),  Wetzlar 
C.  Reichert,  Bennogasse,  26,  Vienna. 

Ernest  Leitz,  Wetzlar, 

F.  W.  Shieck,  Hallesche  Strasse,  14,  Berlin,  S.W. 
Nachet  et  Fils,  Rue  St.  Severin,  17,  Paris. 

C.  Yerick,  Rue  des  Ecoles,  Paris. 


Microtomes. 

To  he  had  from  most  of  the  above  Firms^  and  also  from — 

Zimniermann,  Albert  Strasse,  Leipzig  (Maker  of  Minot’s  Microtome). 

R.  Jung,  Heidelberg  (Maker  of  Tlioma’s  Microtome). 

Schanze,  Pathologisches  Institut,  Liebig  Strasse,  Leipzig. 

W.  Hume,  Lothian  Street,  Edinburgh. 

A.  Fraser,  Lothian  Street,  Edinburgh  (Maker  of  Cathcart’s  Microtome). 
J.  Gardner,  Teviot  Place,  Edinburgh  (Maker  of  Rutherford’s  Microtome). 
Cambridge  Scientific  Instrument  Co. 

Kanthack,  Golden  Square,  off  Regent  Street,  London. 


Chemicals  and  Histological  Heagents. 

Hopkin  & Williams,  16  Cross  Street,  Hatton  Garden,  London,  E.C. 
R.  & J.  Beck,  London. 

Baker,  244  High  Holborn,  London. 

Southall  Brothers  k Barclay,  Dalton  Street,  Birmingham. 

Dr.  Georg  Griibler,  Bayersclie  Strasse,  12,  Leipzig. 


D. — Weights  and  Measures,  Equivalents. 


Centigram  .... 
Decigram  .... 
Gram  ..... 
Kilogram  .... 
I fluid  ounce 
I fluid  drachm 

I inch 

I foot 

ic  00  micros  (yu)  . 

10  millimetres  (mm.)  . 

100  centimetres  (cm.  or  ctm.) 

==  0.0CXD039  inch 
I cm.  ..... 

I metre  .... 


= ,154  English  grains. 

= 1-543 

= 15-432 

= 2.2  lbs.  (avoird.). 

= 28  cubic  centimetre.s. 

= 3*9  >)  it 

= 2.539  centimetres. 

= 3.047  decimetres. 

= I miilimetre. 

==  I centimetre. 

= I metre  (unit  of  length). 

= ^3^J^th  inch  (approximately), 
= 0.3937  inch. 

= 39-3704  inches. 


IITDEX 


Abbe’s  condenser,  lo. 

Absolute  alcohol,  28. 

Absorption  of  fat,  283. 
Acid-alcohol,  65. 

Acid-fuchsin,  74,  154. 

Acids,  30. 

Adenoid  reticulum,  236, 

tissue,  172,  398. 

Adipose  tissue,  168,  398,  401. 
Agininated  follicles,  274. 
Air-bubbles,  loi, 

Albo-carbo  light,  23. 

Albuminous  glands,  258. 

Alcohol,  27. 

dilute,  25. 

Alkalies,  93. 

Alum  carmine,  65. 

Ammoniacal  carmine,  63. 
Ammonium  bichromate,  25,  29. 

chromate,  25,  29. 

Amoeboid  movement,  ill,  117, 
Angle  of  aperture,  13. 
Aniline-blue,  73. 

blue-black,  76. 

dyes,  72. 

oil,  73. 

Anodon’s  muscle,  201. 

Aorta,  226. 

Apochromatic  lenses,  14. 
Aponeurosis,  397. 

Aqueous  humour,  24. 

Arachnoid,  328. 

Areolar  tissue,  159,  161,  397,  400. 
Arrector  pili,  322. 

Arsenic  acid,  37. 

Arteriole,  2290 
Artery,  223. 

elastic  fibres  in,  233. 

endothelium  of,  228. 

Articular  cartilage,  1 50. 

Atrophic  fat-cells,  171. 


A uerbach’s  plexus,  277, 
Axis-cylinder,  206,  21 1. 

Bacteria,  104. 

Balsam,  82,  85,  87. 

Baryta- water,  26. 

Basement  membrane,  31 1. 

Bayerl’s  fluid,  36. 

Benzo-azurin,  13 1. 

Bile-ducts,  290. 

auto-injection  of,  291. 

Bismarck  brown,  75. 

Bladder,  313. 

cells  of,  190,  313. 

frog’s,  134,  190. 

crayfish’s,  115. 

Blood,  colourless  corpuscles  of,  no, 
1 1 7,  122,  402. 

circulation  of,  231. 

division  of,  113. 

effects  of  reagents  on,  1 12,  1 14. 

feeding  of,  113. 

colourless  corpuscles  of,  glycogen 

in,  1 13. 

migration  of,  113. 

Blood-corpuscles,  400. 

amphibians,  106. 

bird,  1 10. 

cover-glass  preparations,  1 14. 

crenation  of,  1 19. 

crystals  from,  120,  402. 

double  staining,  114. 

enumeration  of,  121. 

fish,  no. 

frog,  106. 

human,  115. 

leuksemic,  121. 

pseudo-membrane,  114. 

tablets,  106,  123. 

Blood,  effect  on,  of  acetic  acid,  107, 
112. 


412 


INDEX. 


Blood  aniline  dyes,  12 1. 

of  boracic  acid,  109. 

of  hydrochloric  acid,  108. 

of  magenta,  109. 

of  osmic  acid,  1 10. 

of  syrup,  108. 

of  tannic  acid,  109. 

, of  water,  108. 

Blood-plates,  106,  123. 
Blood-serum,  24. 

Blood-vessels,  223. 

injection  of,  230. 

development  of,  228. 

Bone,  174. 

blood-vessels  of,  179,  181. 

cancellated,  180. 

corpuscles,  177. 

decalcified,  177,  181. 

— — development  of,  182. 

marrow  of,  186. 

perforating  fibres,  178. 

polarised  light,  1 81. 

Borax-carmine,  64. 

Boveri’s  fluid,  213, 

Bowman’s  glands,  375. 

Bronchus,  297, 

Brownian  movement,  100. 
Brunner’s  glands,  278. 

Cabinet,  2. 

Calcified  cartilage,  183. 

Caliciform  cells,  273. 

Camera  lucida,  17. 

Abbe’s,  17, 

Chevalier’s,  18. 

Malassez’s,  18. 

Camera,  Zeiss’s,  17. 

Camera  obscura  shade,  23. 
Canada  balsam,  85. 

Capillaries,  224,  231. 

Capillary  attraction  method,  240. 
Carbolic  acid  and  xylol,  83. 
Cardiac  glands,  267. 

muscle,  199. 

Carmine,  63,  64. 

acid-cliloral,  282. 

and  Dahlia  fluid,  67. 

Frey’s,  641. 

Carter’s  injection,  89. 

Cartilage,  146. 

articular,  150. 

cellular,  146. 

costal,  148. 

cuttlefish,  151. 

encrusting,  184. 

• epiphysial,  184. 


Cartilage,  fibrous,  15 1. 

hyaline,  147,  397. 

parenchymatous,  146. 

transition,  155. 

Caustic  potash,  25. 

Cayal’s  methods,  222. 

Cedar-wood  oil,  83, 

Cell,  animal,  141. 

Celloidin,  45. 

Cell-spaces,  162,  168. 

of  cornea,  361. 

Cellular  cartilage,  146. 

Cement  of  tooth,  251. 

Central  nervous  system,  staining  of, 

338. 

freezing  method,  339. 

Golgi’s  methods,  344. 

Pal’s  method,  343. 

Yessale’s  method,  344. 

Weigert’s  method,  338. 

Weigert-Pal  method,  340. 

Central  tendon,  129,  237. 

Centrifuge,  94. 

Cerebellum,  349. 

blood-vessels  of,  351. 

Golgi’s  method,  355. 

Cerebrum,  351. 

blood-vessels  of,  355. 

GolgFs  method,  357. 

Weigert’s  method,  357. 

Ceruminous  glands,  371. 
Chalice-cells,  139,  273. 

Choroid,  362. 

pigment-cells  of,  363. 

Chromic  acid,  25,  28. 

and  nitric  acid,  36. 

Chromo-ace  tic  acid,  31. 

aceto-osmic  acid,  32. 

-formic  acid,  31. 

— — -osmic  acid,  37. 

Ciliary  muscle,  363. 

motion,  135. 

effects  of  reagents  on,  136. 

processes,  363. 

Ciliated  epithelium,  135. 

isolated  cells,  138. 

Circulation  of  blood,  231. 

in  frog’s  tongue,  234. 

Circumvallate  papillae,  247. 
Clarifying  reagents,  82. 

Clarke’s  column,  330. 

Clasmatocytes,  162. 

Cloves,  oil  of,  83. 

Coarsely-granular  cells,  162. 
Cochineal,  67. 
j Cochlea,  371. 


INDEX. 


413 


Cockroach,  salivary  glands  of,  265. 
Cohnheim’s  areas,  200. 

Collateral  fibres,  346. 

Collodion,  21 1. 

Colostrum,  395. 

Columnar  epithelium,  13 1. 
Condenser,  lo. 

Cones,  368. 

Connective  tissues,  156. 

Convoluted  tubules,  305. 

Copper  acetate,  338. 

Cornea,  358. 

cell- spaces  of,  361. 

corpuscles,  359,  396. 

fibrils  of,  369. 

lymph -spaces,  396. 

nerves  of,  359. 

Corrosive  sublimate,  33. 

Cortex  cerebri,  352. 

Cortical  arteries,  355. 

Corti’s  organ,  373. 

Costal  cartilage,  148. 

Cotton  fibres,  102. 

Cover-glasses,  i. 

to  clean,  98. 

Cover-glass  tester,  23. 

Crab’s  muscle,  196. 

Crayfish  blood,  115. 

Crenation,  119. 

Crista  acustica,  374. 

Creosote,  83, 

Crusta  petrosa,  251. 

Cutting  sections  in  series,  53,  60. 
Cuttlefish  cartilage,  151. 


Dahlia,  73. 

Dammar  lac,  86. 

Decalcifying  fluids,  36. 
Decussation  of  pyramids,  347. 
Demilunes,  258,  260,  265. 
Dentine,  251. 

Descemet’s  membrane,  359. 
Diaphragm,  129,  237,  401. 
Diaphragms,  7. 

Digestion  methods,  26. 

Dilute  alcohol,  25. 

Dissecting  case,  2. 

■ microscopes,  22. 

Dissociating  fluids,  24. 

Dogiel’s  method,  167. 

Drawing  materials,  3. 

Dry  c >ver-glass  preparations,  1 14. 
Duck’s  bill,  378. 

Ductule,  257. 

Dura  mater,  328. 


Ear,  371. 

cartilage,  1 54. 

Ebner’s  fluid,  37. 

Ehrlich-Biondi  fluid,  81,  140. 
Ehrlich’s  hsematoxylin,  69. 

Eimer,  organ  of,  381. 

Elastic  fibres,  157,  397. 

Herxheimer’s  method,  16 1. 

Martinotti’s  reaction,  161. 

Embedding,  40. 

boxes,  45. 

# in  celloidin,  45. 

in  gum,  41. 

in  paraffin,  41,  44. 

interstitial,  41. 

Enamel,  251. 

End-bulbs,  378. 

Endocardium,  223. 

Endothelium,  129,  398. 

of  arteries,  228. 

End-plates,  219. 

Eosin,  72. 

Eosin-haematoxylin,  70. 
Eosinophilous  cells,  122. 

Epidermis  of  man,  317,  325. 

of  newt,  126. 

Epididymis,  387. 

Epiglottis,  153. 

Epiphysis,  184. 

Epithelial  cells,  fibrillation  of,  327. 
Epithelium,  124,  398. 

ciliated,  135. 

-columnar,  13 1. 

germinating,  239. 

glandular,  133. 

secretory,  133. 

squamous,  124. 

transitional,  133. 

Erlicki’s  fluid,  29. 

Eternod’s  rings,  77. 

Eye,  358. 

blood-vessels  of,  369. 

triton’s,  370. 

Eyelid,  3690 

Fallopian  tube,  390. 

Tarrant’s  solution,  85. 

Fat-cell,  169. 

absorption  of,  283. 

action  of  reagents  on,  169. 

■ atrophic,  171. 

development  of,  1 7 1. 

Fenestrated  membranes,  158,  160, 
227. 

Fibres  of  Tomes,  253. 

Fibrin,  119,  123. 


414 


INDEX. 


Fibro-cartilages,  1 51. 

Filiform  papillae,  246, 

Fixatives,  60. 

Fixing  fluids,  27. 

Flemming’s  fluid,  32. 

Fol’s  solution,  32. 

Formatio  reticularis,  348. 

Free  nerve-endings,  376. 

Freezing  fluid,  50. 

Fresh  tissues,  examination  of,  93,  396. 
Frog’s  bladder,  1 34. 

heart,  nerve-cells  of,  233. 

tongue,  140. 

Frommann’s  lines,  207,  21 1. 

Fuchsin,  74. 

Fundus  glands,  268. 

Fungiform  papillae,  247. 

Gamboge,  ioo. 

Ganglion,  spinal,  214. 

sympathetic,  216. 

Gasserian  ganglion,  215. 

Gastric  gland-cells,  268. 

Gaule’s  method,  61. 

Genital  corpuscles,  378. 

Gentian  violet,  73* 

Germinal  epithelium,  389. 
Germinating  epithelium,  239. 
Gland-ducts,  265. 

Glandular  epithelium,  133. 

Glia-cells,  343. 

Glisson’s  capsule,  285. 

Glycerine,  85. 

jelly,  85. 

Glycogen,  293. 

Goblet-cells,  138,  140,  273. 

Gold  chloride,  78. 

Golgi’s  methods,  78,  220,  344. 

■ slow  method,  344. 

sublimate  method,  345. 

rapid  method,  345. 

for  retina,  369. 

Goll’s  column,  330. 

Graafian  follicles,  389. 

Gram’s  method,  105. 

Gran  dry’s  corpuscles,  377. 

Granular  cells,  156,  293,  401. 

Guanin  cells,  173. 

Gustatory  cells,  250. 

Ham-alum,  71. 

Hamatein,  71. 

Hsematoxylin,  68,  71. 

acid,  69. 

• Bohmer’s,  68. 

Delafield’s,  69. 


Hsematoxylin,  Ehrlich’s,  69. 

eosin,  70, 

glycerine,  70. 

Hamilton’s,  69. 

Heidenhain’s,  70. 

Kleinenberg’s,  69. 

Kultschitzky’s,  340. 

nucleus-staining,  69. 

Weigert’s,  338. 

Haemin,  126. 

Haemoglobin,  120. 

Haemolymph,  1 15. 

Hair,-  human,  323. 

blood-pigment  in,  327. 

development  of,  327. 

elements  of,  324. 

rabbit’s,  324. 

Hair-follicles,  321. 

— — coverings  of,  321. 

development  of,  327. 

double-staining  of,  327. 

Hairs,  tactile,  327. 

Half-drying  method,  159. 
Hamilton’s  haematoxylin,  69. 
Hardening  fluids,  27,  33. 

Hard  palate,  126. 

Hartnack’s  dry  lenses,  408. 
Hassall’s  corpuscles,  242. 

Hayem’s  fluid,  122. 

Heart,  223. 

valves,  225. 

Heidenhain’s  method,  70,  259,  282. 
Henle’s  tubule,  305. 

Herbst’s  corpuscles,  379. 

Hermann’s  fluid,  384. 

Herxheimer’s  method,  16 1,  326. 
Horny  epidermis,  127. 

Hot  stage,  117. 

Howship’s  lacunae,  184. 

Hyaline  cartilage,  147. 

Hypodermic  syringe,  161. 
Hypophysis  cerebri,  357. 

Illumination,  artificial,  21. 

direct  and  oblique,  7. 

Immersion  lenses,  12. 

Impregnacion  doble,  222,  346. 
Incremental  lines,  252. 
Indigo-carmine,  67. 

Indirect  cell-division,  14 1. 
Inflammation,  233. 

Injection  mass,  89. 

methods  of,  91. 

Intercellular  bridges,  127. 

channels,  127. 

Intercostal  nerve,  207. 


INDEX. 


415 


Interglobular  spaces,  252. 
Interlobular  artery,  310. 
Intervertebral  disc,  151. 

Intestinal  glands,  283. 

Intestine,  large,  280. 

small,  272. 

Iodine” green,  74. 

Iodised  serum,  25. 

Iris,  363. 

Irregular  tubules,  305. 

Irrigation,  107. 

Kakyokinesis,  141,  145. 

Kidney,  303. 

blood-vessels  of,  306. 

convoluted  tubules,  308. 

fresh,  31 1. 

glomerulus,  307. 

injected,  309. 

irregular  tubules,  308. 

isolated  tubules,  31 1, 

medullary  ray,  309. 

Kleinenberg’s  haematoxylin,  69. 

fluid,  30. 

Klein’s  fluid,  29. 

Kochs- Wolz  lamp,  22. 

Kronecker’s  fluid,  24. 

Kiihne’s  method,  21 1. 
Kultschitzky’s  haematoxylin,  340. 
method,  344. 

Labels,  4. 

Lachrymal  gland,  371. 

Lacteal,  273,  282. 

Landois’  fluid,  26. 

Large  intestine,  280. 

Leitz’s  lenses,  409. 

Lens,  crystalline,  364. 

epithelium  of,  369. 

Lenses,  apochromatic,  15. 

dry,  1 1. 

immersion,  ii. 

Leucocytes,  122,  402,  403. 
Leukaemia,  121. 

Lieberkiihn’s  glands,  273,  2S0. 

mitosis  in,  283. 

Ligamentum  nuchae,  157. 

Linen  fibres,  102. 

Lithium  carmine,  65. 

Liver,  285. 

bile  capillaries  of,  291,  292 

bile  ducts,  290. 

blood-vessels  of,  285,  289. 

cells,  133. 

connective  tissue  of,  292. 

frog’s,  288. 


Liver,  glycogen  in,  293. 

granular  cells  of,  293. 

human,  288. 

iron  in,  293. 

methods  for,  286. 

pigment  in,  293. 

pig’s,  286. 

rabbit’s,  288. 

Lotfler’s  blue,  93. 

Logwood,  71. 

Lugol’s  solution,  93. 

Lungs,  294. 

blood-vessels  of,  294. 

dried,  300. 

elastic  fibres  in,  300,  302. 

foetal,  3CX). 

fresh,  300. 

frog’s,  302. 

newt’s,  302. 

Lymph,  112. 

channels,  237. 

gland,  234,  235,  245. 

Lymphatics,  234. 

Macrophages,  285. 

Macula  lutea,  370. 

Magenta,  74. 

Magnifying  power,  19. 

powers  of  objectives,  408. 

Malpighian  capsule,  304. 

pyramid,  303. 

Mammary  gland,  393. 

active,  395. 

Marchi’s  method,  212,  347. 
Margarine  crystals,  170. 

Marrow,  186,  401. 

Martinotti’s  methods,  161. 
Mastzellen,  67,  156,  162,  401. 

May’s  methods,  219. 

Mayer’s  embedding  bath,  37. 
Measures,  410. 

Medulla  oblongata,  347. 

olivary  bodies,  348. 

Medullary  ray,  303. 

Medullated  nerve-fibres,  202,  399. 
Meissner’s  corpuscles,  380. 

plexus,  277. 

Membrana  tympani,  371. 

Mercuric  chloride,  33. 

r method  for  nervous  system,  345. 

Merkel’s  corpuscles,  377. 

Methylated  spirit,  28. 
Methylene-blue,  73,  131,  192,  222^ 
370, 284. 

Methyl -green,  74. 

Methyl-mixture,  26. 


4i6 


INDEX. 


Methyl-violet,  73. 

Micrococci,  104. 

Micrometer,  20. 

Micro-organisms,  103. 

Microphages,  285. 

Microscope,  5. 

choice  of,  15. 

dissecting,  23- 

illumination  of,  7. 

lamp,  21, 

magnifying  power  of,  19. 

makers  of,  409. 

parts  of,  1 5. 

Microtomes,  49. 

Cambridge,  53. 

Cathcart’s,  53. 

Jung’s,  56. 

makers  of,  410. 

Malassez’s,  56. 

Minot’s,  55. 

Ranvier’s,  59. 

Roy’s,  53. 

Rutherford’s,  50. 

Swift’s,  59. 

Thoma’s,  56. 

Williams’,  58. 

Migratory  cells,  157. 

Milk,  98. 

Mitosis,  14 1,  145. 

Mounting  block,  89. 

fluids  and  methods,  85. 

Mole’s  nose,  381. 

Mucigen,  139,  260. 

Muco-salivary  glands,  257,  262,  264. 
Mucous  cells,  265. 

glands,  256,  258. 

tissue,  17 1,  174, 

Muller’s  fluid,  29. 

and  spirit,  29. 

Multipolar  nerve-cells,  217,  343. 
Muscle,  189,  398. 

striped,  189,  398. 

Myelin,  205. 

drops,  205. 

Myeloplaxes,  187. 

Myocardium,  223. 

^N'atl,  326. 

double-stained,  327. 

Needles,  2. 

Nerve-cells,  213,  399. 

cover-glass  } (reparations  of,  21 8. 

crayfish,  223. 

in  frog’s  heart,  221,  233. 

multipolar,  217,  343. 

pyriform,  221. 


Nerv^e-cells,  spinal  cord,  217. 

sympathetic,  217,  222. 

transverse  markings  on,  207. 

Nerve-endings,  376. 

Nerve-plexuses,  276, 

Nerve-fibres,  202. 

axis-cylinder  of,  205,  208,  211. 

degeneration  of,  213. 

in  osmic  acid,  206,  21 1. 

intercostal,  207. 

living,  212. 

— — medullated,  202,  399. 

non-medullated,  203,  209. 

size  of,  212. 

spinal  cord,  213. 

to  muscle,  219. 

transverse  markings  on,  207. 

Nerve-plexus  in  intestine,  277. 
Nerve-trunks,  203. 

Neuroglia,  341,  343. 

Neurokeratin,  210. 

Newt’s  cartilage,  145. 

Nitric  acid,  31. 

N odes  of  Ranvier,  202. 
Non-medullated  nerve-fibres,  203. 
Non-striped  muscle,  189. 

cement  of,  19 1. 

grooving  on,  193. 

plexus  in,  192. 

Normal  fluids,  24. 

saline,  24. 

Nose,  374,  376. 

Nuclear  stains,  63. 

Objectives,  9. 

Obreggia’s  method,  404. 

Oculars,  5,  10. 

Odontoblasts,  253. 

(Esophagus,  255. 

and  stomach,  271, 

Oikoid,  109. 

Olfactory  bulb,  376. 

cells,  37S. 

Omentum,  129. 

Onion,  103. 

Opaque  injections,  289. 

Optic  nerve,  368. 

entrance  of,  370. 

Origanum  oil,  83. 

Osmic  acid,  25,  32. 
Osmico-bichromaie  mixture,  345. 
Ossification,  182. 

Osteoblasts,  178. 

Osteoclasts,  183. 

Ovary,  388, 

Ovum,  390. 


INDEX. 


41.7 


Pacinian  corpuscle,  378. 
Palate,  hard,  126. 

glands  of,  251. 

soft,  250. 

Palm  of  hand,  317. 

Pal’s  method,  343. 

Pancreas,  262,  404. 

cells  of,  265. 

nerves  of,  266. 

Papillae  foliatae,  249. 

of  skin,  315. 

of  tongue,  246. 

Paraffin,  41. 

Penicillium,  103. 

Penis,  313. 

Perenyi’s  fluid,  31, 

Perforating  fibres,  178. 
Pericardium,  223. 
Perioesophageal  membrane,  163. 
Periodontal  membrane,  252. 
Periosteum,  177. 

Peyer’s  patch,  272,  274,  284. 
Phagocytosis,  284. 
Phenylene-brown,  75. 
Phloroglucin,  37. 

Photophore,  27. 

Pia  mater,  228. 

Picric  acid,  30,  36. 
Picrin-glycerine,  192. 
Picro-carniine,  66,  80. 
Picro-glycerine  mixture,  192. 
Picro-lithium  carmine,  66. 
Picro-nitric  acid,  31. 
Picro-sulphuric  acid,  30. 
Pigment-cells,  173,  397. 

choroid,  363. 

Pipettes,  4. 

Pituitary  body,  357. 

Placenta,  395. 

injected,  396. 

Plasma-cells,  156. 

Polaiiscope,  200. 

Potassic  bichromate,  25,  29. 
Potato  starch,  100. 

Preparation  of  tissues,  38,  396. 
Prickle  cells,.  127,  318. 

Pulp  cavity,  253. 

Purkinje’s  cells,  351. 

fibres,  225. 

Pyloric  glands,  270. 

Py loro-duodenal  region,  271, 
Pyriform  nerve-cells,  221. 

Rabl’s  fluid,  31. 

Ranvier’s  crosses,  207,  21 1. 

fluid,  25. 

37 


I Ranvier’s  nodes,  206, 

I Razor,  3. 

I Rectified  spirit,  28. 

Red  marrow,  187. 

Respiratory  organs,  295. 

Retina,  365,  370,  399. 

cones  of,  367. 

frog’s,  368. 

Golgi’s  methods  for,  369. 

macula  lutea,  370. 

Retro-lingual  membrane,  201. 
Rice-starch,  100. 

Ripart  and  Petit’s  fluid,  24. 
Rosanilin,  74. 

Safranin,  75. 

Salivary  corpuscles,  125. 

glands,  256,  404. 

of  cockroach,  265. 

Sarcolemma,  193. 

Sarcostyles,  198. 

Scalp,  323. 

Schiefferdecker’s  fluid,  26, 
Schizomycetes,  105. 

Schreger’s  lines,  252. 

Sebaceous  glands,  323. 

Section  cutting,  48. 

flatteners,  59. 

in  series,  60. 

lifter,  3. 

to  place  on  slide,  86. 

Semicircular  canals,  374. 
Semidesiccation  method,  159. 
Seminiferous  tubules,  382. 
Sensory  nerve-terminations,  376. 
Septum  cisteriise,  238. 

Serial  sections,  60. 

Serous  fluids,  113. 

glands,  257,  261. 

nerves  in,  265. 

Serum,  action  of,  124. 

and  osmic  acid,  78. 

Sharpey’s  fibres,  178. 

Silver  lines,  129. 

nitrate,  76. 

Skin,  315. 

blood-vessels  of,  325. 

cutis  vera,  315. 

elastic  fibres  in,  326. 

epidermis  of,  325. 

finger,  317. 

foetal,  320. 

injected,  325. 

negro’s,  320. 

nerves  of,  377. 

sebaceous  glands  of,  322. 

2 D 


4i8 


INDEX. 


Skin,  sweat-glands  of,  319,  327. 

touch-corpuscles  ot,  320. 

to  clean,  98. 

Slides,  I. 

Small  intestine,  272. 

absori>tion  of  fat,  283. 

blood-vessels  of,  275. 

nerve-plexuses  of,  276. 

nerves  of,  284. 

Soft  palate,  250 
Solitary  follicles,  275,  280. 
Spermatogenesis,  383,  385. 
Spermatozoa,  386. 

cover-glass  preparations,  387. 

frog’s,  386. 

human,  387. 

newt’s,  386. 

Spiller’s  purple,  74. 

Spinal  cord,  328. 

cells  of,  217,  329. 

collateral  fibres,  346. 

columns  of,  330. 

commissures  of,  329. 

cornua,  329, 

degeneration  of,  332,  347. 

dry  preparation  of,  342. 

fissures  of,  328. 

ganglia,  213. 

grey  matter  of,  334. 

human,  337. 

longitudinal  section,  338. 

nerve-cdls,  343. 

nerve-fibres  of,  341. 

neuroglia  of,  341. 

staining  of,  341. 

substantia  gelatinosa,  329. 

tracts  in,  542, 

white  matter  of,  335. 

Spleen,  242. 

injected,  245, 

Squames,  125. 

Staining  in  bulk,  44. 

general  remarks,  8i. 

multiple,  80. 

reagents,  63. 

Starch,  potato,  100. 

rice,  100. 

Stomach,  266. 

blood-vessels  of,  271 

■ cardiac  end,  267. 

double  staining  of,  271. 

methods  for,  267. 

nerves  of,  284. 

pyloric  end,  270. 

Stomata,  239. 

Stratified  epithelium,  126. 


Striped  muscle,  193,  398. 

Anodon’s,  201. 

cardiac,  199. 

crab’s,  196,  200. 

discs,  195. 

fibrillse  of,  195, 

frozen,  200. 

injected,  198. 

isolated  fibres,  194. 

living,  200. 

red,  199. 

sarcolemma,  193. 

tendon  of,  196. 

Sublingual  gland,  259. 
Submaxillary  glands,  25Q.  262,  264. 
Suprarenal  capsule,  314. 

nerves  of,  315. 

Sweat-glands,  319,  327. 
Sympathetic  nerve-fibres,  209. 

ganglia,  216. 

nerve-cells,  222. 

Cayal’s  method,  222. 

Syringes,  91, 

Tactile  cells,  377,  381. 

■ — - disc,  377. 

hairs,  327. 

Taste-buds,  249. 

Teasing,  26. 

Tendon,  163,  397. 

nerves  in,  221. 

Terminal  corpuscles,  377. 

Testis,  387. 

Thymus  gland,  241. 

Thyroid  gland,  301. 

Tizzoni’s  reaction,  293. 

Tongue,  246, 

circulation  in,  234. 

frog’s,  140. 

■ — — gland  of,  251. 

injected,  248. 

nerves  of,  251. 

papillaj  of,  246. 

Tonsils,  239. 

Tooth,  251. 

development  of,  253. 

softened,  252. 

’rouch-corpuscle,  380. 

Trachea,  294. 

’fransitional  epithelium,  133. 
Tubules  isolated  of  kidney,  3 II. 

Umbilical  cord,  395. 

Unna’s  method,  327. 

Ureter,  312. 

Uterus,  392, 


Valentin’s  knife,  49. 

Valves  of  heart,  225. 

Vas  deferens,  388. 

Vegetable  cells,  103. 

Veins,  224,  230. 

Vermiform  appendix,  281,  284. 
Vessale’s  method,  344. 

Vesuvin,  76. 

Villus  of  intestine,  272,  280,  282. 

injected,  275. 

Volkmann’s  canals,  176. 

Von  Ebner’s  fluid,  37. 

Wagner’s  touch-corpuscles,  380. 
Warm  stages,  117. 

Weigert’s  hsematoxylin,  338. 

method  for  staining  central  ner- 
vous system,  338. 


INDEX. 

Weigert-Pal  method,  340. 
Weights,  410. 

Welsbach  light,  22. 
Westphal’s  fluid,  67. 
Wharton’s  jelly,  171. 
White  fibro-cartilage,  152. 

zinc  cement,  89. 

Wool,  102,  324. 

W orks  of  reference,  406. 

Xylol,  83. 

balsam,  86. 

Yeast,  104. 

Zeiss’s  lenses,  409. 

Zinc  cement,  89. 

Zooid,  109. 


410 


Iri 


) 


Catalogue  No.  8. 


June,  1897. 


CLASSIFIED  SUBJECT 
CATALOGUE 

OF 

MEDICAL  BOOKS 

AND 

Books  on  Medicine,  Dentistry,  Pharmacy, 
Chemistry,  Hygiene,  Etc.,  Etc., 

PUBLISHED  BY 

P.  Blakiston,  Son  & Co., 

Medical  Publishers  and  Booksellers, 

1012  WALNUT  STREET,  PHILADELPHIA. 


SPECIAL  NOTE. — The  prices  given  in  this  catalogue  are 
absolutely  net,  no  discount  will  be  allowed  retail  purchasers 
under  any  consideration.  This  rule  has  been  established  in 
order  that  everyone  will  be  treated  alike,  a general  reduction 
in  former  prices  having  been  made  to  meet  previous  retail  dis- 
counts. Upon  receipt  of  the  advertised  price  any  book  will 
be  forwarded  by  mail  or  express,  all  charges  prepaid. 


We  keep  a large  stock  of  Miscellaneous  Books,  not  on  this 
catalogue,  relating  to  Medicine  and  Allied  Sciences,  pub 
lished  in  this  country  and  abroad.  Inquiries  in  regard  to 
prices,  date  of  edition,  etc.,  will  receive  prompt  attention. 


Special  Catalogues  of  Books  on  Pharmacy,  Dentistry, 
Chemistry,  Hygiene,  and  Nursing  will  be  sent  free  upon 
application. 

^SEE  NEXT  PAGE  FOR  SUBJECT  INDEX. 
Gonld^s  Dictionaries^  Page  8, 


SUBJECT  INDEX 


4®=*  Any  books  not  on  this  Catalogue  we  will  furnish  a price 
for  upon  application. 


SUBJECT.  PAGE 

Alimentary  Canal  (see  Surgery)  19 

Anatomy 3 

Anesthetics 3 

Autopsies  (see  Pathology) 16 

Bandaging  (see  Surgery) 19 

Brain  4 

Chemistry 4 

Children,  Diseases  of 6 

Clinical  Charts 6 

Compends 22,  23 

Consumption  (see  Lungs) 12 

Deformities 7 

Dentistry 7 

Diagnosis 17 

Diagrams  (see  Anatomy,  page 
3,  and  Obstetrics,  page  16). 

Dictionaries 8 

Diet  and  Food  (see  Miscella- 
neous)   14 

Dissectors 3 

Domestic  Medicine 10 

Ear 8 

Electricity 9 

Emergencies  (see  Surgery) 19 

Eye 9 

Fevers 9 

Gout  10 

Gynecology 21 

Headaches 10 

Heart 10 

Histology 10 

Hospitals  (see  Hygiene) 11 

Hygiene ii 

Insanity  4 

Journals  ii 

Kidneys  12 

Latin,  Medical  (see  Miscella- 
neous and  Pharmacy) 14,  16 

Lungs 12 

Massage 12 

Materia  Medica 12 

Medical  Jurisprudence 13 

Microscopy  13 

Milk  Analysis  (see  Chemistry)  4 


SUBJECT.  PAGE 

Miscellaneous  14 

Nervous  Diseases  14 

Nose 20 

Nursing 15 

Obstetrics 16 

Ophthalmology 9 

Osteology  (see  Anatomy) 3 

Pathology 16 

Pharmacy 16 

Physical  Diagnosis 17 

Physical  Training  (see  Miscel- 
laneous)  14 

Physiology  18 

Poisons  (see  Toxicology) 13 

Popular  Medicine 10 

Practice  of  Medicine 18 

Prescription  Books 18 

Railroad  Inj'uries  (see  Nervous 

Diseases) 14 

Refraction  (see  Eye) 9 

Rheumatism  10 

Sanitary  Science ii 

Skin 19 

Spectacles  (see  Eye) 9 

Spine  (see  Nervous  Diseases)  14 
Stomach  (see Miscellaneous)...  14 

Students*  Compends 22,  23 

Surgery  and  Surg.  Diseases...  19 

Syphilis 21 

Technological  Books 4 

Temperature  Charts 6 

Therapeutics 12 

Throat  20 

Toxicology 13 

U.  S.  Pharmacopoeia 16 

Urinary  Organs 20 

Urine 20 

Venereal  Diseases 21 

Veterinary  Medicine 21 

Visiting  Lists,  Physicians’. 

{Send  for  Special  Circular.) 
Water  Analysis  (see  Chemis- 
try)  II 

Women,  Diseases  of. 21 


The  prices  as  given  in  this  Catalogue  are  net.  Cloth 
binding,  unless  otherwise  specified.  No  discount  can  be 
allowed  under  any  circumstances.  Any  book  will  be  sent, 
postpaid,  upon  receipt  of  advertised  price. 


SUBJECT  CATALOGUE  OF  MEDICAL  BOOKS. 


3 


4®=“  All  books  are  hound  in  cloth f unless  otherwise  speci- 
fied. All  prices  a/re  net. 

ANATOMY. 

MORRIS.  Text-Book  of  Anatomy.  791  Ulus.,  214  of  which  are 
printed  in  colors.  Clo.,  $6.00  ] Lea.,  $7.00;  Half  Russia,  $8.00. 

Taken  as  a whole,  we  have  no  hesitation  in  according  very  high 
praise  to  this  work.  It  will  rank,  we  believe,  with  the  leading  Anato- 
mies. The  illustrations  are  handsome  and  the  printing  is  good.*’— 
Boston  Medical  and  Surgical  Journal. 

Handsome  Circular  of  Morris,  with  sample  pages  and  colored  illus- 
trations, will  be  sent  free  to  any  address. 

CAMPBELL.  Outlines  for  Dissection.  Prepared  for  Use  with 
“ Morris's  Anatomy"  by  the  Demonstrator  of  Anatomy  at  the  Uni- 
versity of  Michigan.  $1.00 

HEATH.  Practical  Anatomy.  A Manual  ot  Dissections.  8th 

Edition.  300  Illustrations.  $4.25 

HOLDEN.  Anatomy.  A Manual  of  the  Dissections  of  the  Human 
Body.  6th  Edition.  Carefully  Revised  by  A.  Hkwson,  m.d..  De- 
monstrator of  Anatomy,  Jefferson  Medical  College,  Philadelphia. 
31 1 Illustrations.  Cloth,  $2.50  ; Oil-Cloth,  ^2.50 ; Leather,  $3.00 
HOLDEN.  Human  Osteology.  Comprising  a Description  of  the 
Bones,  with  Colored  Delineations  of  the  Attachments  of  the  Muscles. 
The  General  and  Microscopical  Structure  of  Bone  and  its  Develop- 
ment. With  Lithographic  Plates  and  numerous  Ulus.  7th  Ed.  $5.25 
HOLDEN.  Landmarks.  Medical  and  Surgical.  4th  Ed.  ^i.oo 
MACALISTER.  Human  Anatomy.  Systematic  and  Topograph- 
ical, including  the  Embryology,  Histology,  and  Morphology  of  Man. 
With  Special  Reference  to  the  Requirements  of  Practical  Surgery  and 
Medicine.  816  Illustrations,  400  of  which  are  original. 

Cloth,  ^5.00;  Leather,  ^6.00 
MARSHALL.  Physiological  Diagrams.  Life  Size,  Colored. 
Eleven  Life-Size  Diagrams  (each  seven  feet  by  three  feet  seven 
inches).  Designed  for  Demonstration  before  the  Class. 

In  Sheets,  Unmounted,  $40.00;  Backed  with  Muslin  and  Mounted 
on  Rollers,  $60.00;  Ditto,  Spring  Rollers,  in  Handsome  Walnut  Wall 
Map  Case  (send  for  special  circular),  $100.00;  Single  Plates — Sheets, 
$5.00;  Mounted,  $7.50.  Explanatory  Key,  .50.  Descriptive  circu- 
lar upon  application. 

POTTER.  Compend  of  Anatomy,  Including  Visceral  Anatomy. 
5th  Edition.  16  Lithographed  Plates  and  117  other  Illustrations. 

.80 ; Interleaved,  $1.25 

WILSON.  Human  Anatomy,  nth  Edition.  429  Illustrations,  26 
Colored  Plates,  and  a Glossary  of  Terms.  $5.00 

WINDLE.  Surface  Anatomy  and  Landmarks.  Colored  and 
other  Illustrations.  Just  Ready.  $1.00 

ANESTHETICS. 

BUXTON.  On  Anesthetics.  2d  Edition.  Illustrated.  $125 
TURNBULL.  Artificial  Anesthesia.  The  Advantages  and 
Accidents  of;  Its  Employment  in  the  Treatment  of  Disease  ; Modes 
of  Administration ; Considering  their  Relative  Risks ; Tests  of 
Purity;  Treatment  of  Asphyxia;  Spasms  of  the  Glottis;  Syncope, 
etc.  4th  Edition,  Revised.  54  Illustrations,  fust  Ready.  $2.50 


4 


SUBJECT  CATALOGUE. 


BRAIN  AND  INSANITY. 

BLACKBURN.  A Manual  of  Autopsies.  Designed  for  the  Use 
of  Hospitals  for  the  Insane  and  other  Public  Institutions.  Ten  full- 
page  Plates  and  other  Illustrations.  $125 

GOWERS.  Diagnosis  of  Diseases  of  the  Brain.  2d  Edition. 

Illustrated.  $i-5o 

HORSLEY.  The  Brain  and  Spinal  Cord.  The  Structure  and 
Functions  of.  Numerous  Illustrations.  $2.50 

HYSLOP.  Mental  Physiology.  Especially  in  Relation  to  Men- 
tal Disorders.  With  Illustrations. 

LEWIS  (BEVAN).  Mental  Diseases.  A Text  Book  Having 
Special  Reference  to  the  Pathological  Aspects  of  Insanity.  18  Litho- 
graphic Plates  and  other  Illustrations.  New  Edition.  In  Press. 
MANN.  Manual  of  Psychological  Medicine  and  Allied 
Nervous  Diseases.  Their  Diagnosis,  Pathology,  Prognosis,  and 
Treatment,  including  their  Medico-Legal  Aspects  ; with  chapter  on 
Expert  Testimony,  and  an  Abstract  of  the  Laws  Relating  to  the 
Insane  in  all  the  States  of  the  Union.  Illustrations  of  Typical  Faces 
of  the  Insane,  Handwriting  of  the  Insane,  and  Micro-photographic 
Sections  of  the  Brain  and  Spinal  Cord.  ^3.00 

REGIS.  Mental  Medicine.  Authorized  Translation  by  H.  M. 

Bannister,  m.d.  $2.00 

STEARNS.  Mental  Diseases.  Designed  especially  for  Medical 
Students  and  General  Practitioners.  With  a Digest  of  Laws  of  the 
various  States  Relating  to  Care  of  Insane.  Illustrated. 

Cloth,  $2.75;  Sheep,  $3.25 
TUKE.  Dictionary  of  Psychological  Medicine.  Giving  the 
Definition,  Etymology,  and  Symptoms  of  the  Terms  used  in  Medical 
Psychology,  with  the  Symptoms,  Pathology,  and  Treatment  of  the 
Recognized  Forms  of  Mental  Disorders,  together  with  the  Law  of 
Lunacy  in  Great  Britain  and  Ireland.  Two  volumes.  ^10.00 

WOOD,  H.  C.  Brain  and  Overwork.  .40 

CHEMISTRY  AND  TECHNOLOGY. 

special  Catalogue  oj  Chemical  Books  sent  free  upon  application. 

ALLEN.  Commercial  Organic  Analysis.  A Treatise  on  the 
Modes  of  Assaying  the  Various  Organic  Chemicals  and  Products 
Employed  in  the  Arts,  Manufactures,  Medicine,  etc.,  with  concise 
methods  for  the  Detection  of  Impurities,  Adulterations,  etc.  2d  Ed. 
Vol.  I,  Vol.  II,  Vol.  Ill,  Part  I.  These  volumes  cannot  be  had. 
Vol.  Ill,  Part  II.  The  Amins.  Pyridin  and  its  Hydrozins  and 
Derivatives.  The  Antipyretics,  etc.  Vegetable  Alkaloids,  Tea, 
Coffee,  Cocoa,  etc.  $4- 50 

Vol.  Ill,  Part  III.  Animal  Bases,  Animal  Acids,  Cyanogen  Com- 
pounds, Proteids,  etc.  $4-5o 

Vol.  Ill,  Part  IV.  The  Proteids  and  Albuminoids.  In  Press. 

ALLEN.  Chemical  Analysis  of  Albuminous  and  Diabetic 
Urine.  Illustrated.  $2.25 

BARTLEY.  Medical  and  Pharmaceutical  Chemistry.  A 
Text-Book  for  Medical,  Dental,  and  Pharmaceutical  Students.  With 
Illustrations,  Glossary,  and  Complete  Index.  4th  Edition,  carefully 
Revised.  Cloth,  $2.75 ; Sheep,  ^3.25 

BLOXAM.  Chemistry,  Inorganic  and  Organic.  With  Experi- 
ments. 8th  Ed.,  Revised.  281  Engravings.  CTo.,  $4.25  ; Lea.,  $5.25 


MEDICAL  BOOKS. 


5 


CALDWELL.  Elements  of  Qualitative  and  Quantitative 
Chemical  Analysis.  3d  Edition,  Revised.  ^i-50 

CAMERON.  Oils  and  Varnishes.  With  Illustrations,  Formulae, 
Tables,  etc.  ^2.25 

CAMERON.  Soap  and  Candles.  54  Illustrations.  $2.00 

CLOWES  AND  COLEMAN.  Elementary  Qualitative  An- 
alysis. Adapted  for  Use  in  the  Laboratories  of  Schools  and  Colleges. 
Illustrated.  $1.00 

GARDNER.  The  Brewer,  Distiller,  and  Wine  Manufac- 
turer. A Hand-Book  for  all  Interested  in  the  Manufacture  and 
Trade  of  Alcohol  and  Its  Compounds.  Illustrated. 

GARDNER.  Bleaching,  Dyeing,  and  Calico  Printing.  With 
P'ormulae.  Illustrated.  $1.50 

GROVES  AND  THORP.  Chemical  Technology.  The  Appli- 
cation of  Chemistry  to  the  Arts  and  Manufactures.  8 Volumes, 
with  numerous  Illustrations. 

Vol.  I.  Fuel  and  Its  Applications.  607  Illustrations  and  4 Plates. 

• Cloth,  ^5,00;  Half  Morocco,  $6.50 

Vol.  II.  Lighting.  Illustrated.  Cloth,  ^4.00;  Half  Morocco,  ^5.50 
Vol.  III.  Lighting — Continued.  In  Press. 

HOLLAND.  The  Urine,  the  Gastric  Contents,  the  Common 
Poisons,  and  the  Milk.  Memoranda,  Chemical  and  Microscopi- 
cal, for  Laboratory  Use.  5th  Ed.  Illustrated  and  interleaved,  $1.00 

LEFFMANN.  Compend  of  Medical  Chemistry,  Inorganic 
and  Organic.  Including  Urine  Analysis.  4th  Edition,  Rewritten. 

.80 ; Interleaved,  ^1.25 

LEFFMANN.  Progressive  Exercises  in  Practical  Chemis- 
try. Illustrated.  2d  Edition.  $1.00 

LEFFMANN.  Analysis  of  Milk  and  Milk  Products.  Arranged 
to  Suit  the  Needs  of  Analytical  Chemists,  Dairymen,  and  Milk  Inspec- 
tors. 2d  Edition.  Enlarged,  Illustrated.  Just  Ready.  ^1-25 

LEFFMANN.  Water  Analysis.  Illustrated.  3d  Edition.  $1.25 

LEFFMANN.  Structural  Formulae  for  the  Use  of  Students. 
Including  180  Structural  and  Stereo-Chemical  Formulae.  i2mo. 
Interleaved.  Just  Ready.  ^i.oo 

MUTER.  Practical  and  Analytical  Chemistry.  4th  Edition. 
Revised  to  meet  the  requirements  of  American  Medical  Colleges  by 
Claude  C.  Hamilton,  m.d.  51  Illustrations.  $1.25 

OVERMAN.  Practical  Mineralogy,  Assaying,  and  Mining. 
With  a Description  of  the  Useful  Minerals,  etc.  nth  Edition.  ^i.oo 

RICHTER.  Inorganic  Chemistry.  4th  American,  from  6th  Ger- 
man Edition.  Authorized  translation  by  Edgar  F.  Smith,  m.a., 
PH.D.  89  Illustrations  and  a Colored  Plate.  $i-75 

RICHTER.  Organic  Chemistry.  3d  American  Edition.  Trans, 
from  the  last  German  by  Edgar  F.  Smith.  Illustrated.  In  Press. 

SMITH.  Electro-Chemical  Analysis.  2d  Edition,  Revised.  28 
Illustrations.  $1.25 

SMITH  AND  KELLER.  Experiments.  Arranged  for  Students 
in  General  Chemistry.  3d  Edition.  Illustrated.  .60 

STAMMER.  Chemical  Problems.  With  Explanations  and  An- 
swers. CQ 


6 


SUBJECT  CATALOGUE. 


SUTTON.  Volumetric  Analysis.  A Systematic  Handbook  for 
the  Quantitative  Estimation  of  Chemical  Substances  by  Measure, 
Applied  to  Liquids,  Solids,  and  Gases.  7th  Edition,  Revised.  112 
Illustrations.  Just  Ready.  $4.50 

SYMONDS.  Manual  of  Chemistry,  for  Medical  Students. 

2d  Edition.  ^2.00 

WATTS.  Organic  Chemistry.  2d  Edition.  By  Wm.  A.  Tildkn, 
D.sc.,F.R.s.  (Being  the  13th  Edition  of  Fowne’s  Organic  Chemistry.) 
Illustrated.  ^2  00 

WATTS.  Inorganic  Chemistry.  Physical  and  Inorganic.  (Being 
the  14th  Edition  of  Fowne's  Physical  and  Inorganic  Chemistry.) 
With  Colored  Plate  of  Spectra  and  other  Illustrations.  ^2.00 

WOODY.  Essentials  of  Chemistry  and  Urinalysis.  4th 
Edition.  Illustrated.  In  Press. 

***  special  Catalogue  of  Books  on  Chemistry  free  ufon  application. 


CHILDREN. 

CAUTLIE.  Feeding  of  Infants  and  Young  Children  by  Nat- 
ural and  Artificial  Methods.  Just  Ready.  $2.00 

HALE.  On  the  Management  of  Children  in  Health  and  Dis- 
ease. .50 

HATFIELD.  Compend  of  Diseases  of  Children.  With  a 
Colored  Plate.  2d  Edition.  .80;  Interleaved,  25 

MEIGS.  Infant  Feeding  and  Milk  Analysis.  The  Examination 
of  Human  and  Cow’s  Milk,  Cream,  Condensed  Milk,  etc.,  and 
Directions  as  to  the  Diet  of  Young  Infents.  .50 

MONEY.  Treatment  of  Diseases  in  Children.  Including  the 
Outlines  of  Diagnosis  and  the  Chief  Pathological  Differences  Between 
Children  and  Adults.  2d  Edition.  ^2.50 

POWER.  Surgical  Diseases  of  Children  and  their  Treat- 
ment by  Modern  Methods.  Illustrated.  $2.50 

STARR,  The  Digestive  Organs  in  Childhood.  The  Diseases  of 
the  Digestive  Organs  in  Infancy  and  Childhood.  With  Chapters  on 
the  Investigation  of  Disease  and  the  Management  of  Children.  2d 
Edition,  Enlarged.  Illustrated  by  two  Colored  Plates  and  numerous 
Wood  Engravings.  $2.00 

STARR.  Hygiene  of  the  Nursery.  Including  the  General  Regi- 
men and  Feeding  of  Infants  and  Children,  and  the  Domestic  Manage- 
ment of  the  Ordinary  Emergencies  of  Early  Life,  Massage,  etc.  6th 
Edition.  25  Illustrations.  Just  Ready.  $1.00 

TAYLOR  AND  WELLS.  The  Diseases  of  Children.  Illus- 
trated. A New  Text-Book.  Nearly  Ready. 


CLINICAL  CHARTS. 

GRIFFITH.  Graphic  Clinical  Chart.  Printed  in  three  colors. 
Sample  copies  free.  Put  up  in  loose  packages  of  fifty,. 50.  Price  to 
Hospitals,  500  copies,  $4.00 ; 1000  copies,  $7.50.  With  name  of 
Hospital  printed  on,  .50  extra. 

TEMPERATURE  CHARTS.  For  Recording  Temperature, 
Respiration,  Pulse,  Day  of  Disease,  Date,  Age,  Sex,  Occu- 
pation, Name,  etc.  Put  up  in  pads  of  fifty.  Each,  .50 


MEDICAL  BOOKS. 


7 


DEFORMITIES. 

REEVES.  Bodily  Deformities  and  Their  Treatment.  A 
Hand-Book  of  Practical  Orthopedics.  228  Illustrations.  $i»75 

HEATH.  Injuries  and  Diseases  of  the  Jaws.  187  Illustrations. 
4th  Edition.  Cloth,  ^4.50 


DENTISTRY. 

special  Catalogue  0/  Dental  Books  sent  free  upon  application. 

BARRETT.  Dental  Surgery  for  General  Practitioners  and 
Students  of  Medicine  and  Dentistry.  Extraction  of  Teeth, 
etc.  3d  Edition.  Illustrated.  Nearly  Ready. 

BLODGETT.  Dental  Pathology.  By  Albert  N.  Blodgett, 
M.D.,  late  Professor  of  Pathology  and  Therapeutics,  Boston  Dental 
College.  33  Illustrations.  $1.25 

FLAGG.  Plastics  and  Plastic  Filling,  as  Pertaining  to  the  Filling 
of  Cavities  in  Teeth  of  all  Grades  of  Structure.  4th  Edition.  $4.00 
FILLEBROWN.  A Text-Book  of  Operative  Dentistry. 
Written  by  invitation  of  the  National  Association  of  Dental  Facul- 
ties. Illustrated.  ^2,25 

GORGAS.  Dental  Medicine.  A Manual  of  Materia  Medica  and 
Therapeutics.  5th  Edition,  Revised.  Cloth,  ^4.00;  Sheep,  $5.00 
HARRIS.  Principles  and  Practice  of  Dentistry.  Including 
Anatomy,  Physiology,  Pathology,  Therapeutics,  Dental  Surgery, 
and  Mechanism.  13th  Edition.  Revised  by  F.  J.  S.  Gorgas,  m.d., 
D.D.s.  1250  Illustrations.  Cloth,  ^6.00;  Leather,  1^7.00 

HARRIS.  Dictionary  of  Dentistry.  Including  Definitions  of  Such 
Words  and  Phrases  of  the  Collateral  Sciences  as  Pertain  to  the  Art  and 
Practice  of  Dentistry.  5th  Edition.  Revised  and  Enlarged  by  Fer- 
dinand F.  S.  Gorgas,  m d.,  d.d.s.  Cloth,  $4.50;  Leather,  ^5.50 
HEATH.  Injuries  and  Diseases  of  the  Jaws.  4th  Edition.  187 
Illustrations.  ^4-5o 

HEATH.  Lectures  on  Certain  Diseases  of  the  Jaws.  64 
Illustrations.  Boards,  .50 

RICHARDSON.  Mechanical  Dentistry.  7th  Edition.  Thor- 
oughly Revised  and  Enlarged  by  Dr.  Geo.  W.  Warren.  691  Illus- 
trations. Just  Ready.  Cloth,  ;^5. 00;  Leather,  ^6.00 

SEWELL.  Dental  Surgery.  Including  Special  Anatomy  and 
Surgery.  3d  Edition,  with  200  Illustrations.  ^2.00 

TAFT.  Operative  Dentistry.  A Practical  Treatise.  4th  Edition. 

100  Illustrations.  Cloth,  $3.00  ; Leather,  ^4.00 

TAFT.  Index  of  Dental  Periodical  Literature.  $2.00 

TALBOT.  Irregularities  of  the  Teeth  and  Their  Treatment. 

2d  Edition.  234  Illustrations.  $3.00 

TOMES.  Dental  Anatomy.  Human  and  Comparative.  235  Illus- 
trations. 4th  Edition.  ^3-5o 

TOMES.  Dental  Surgery.  3d  Edition.  292  Illustrations.  $4.00 
WARREN.  Compend  of  Dental  Pathology  and  Dental  Medi- 
cine. With  a Chapter  on  Emergencies.  Illustrated. 

.80;  Interleaved,  $1.25 

WARREN.  Dental  Prosthesis  and  Metallurgy.  129  Ills.  $1.25 
WHITE.  The  Mouth  and  Teeth.  Illustrated.  .40 

%*  Special  Catalogue  Dental  Books  free  upon  application. 


8 


SUBJECT  CATALOGUE. 


DICTIONARIES. 

GOULD.  The  Illustrated  Dictionary  of  Medicine,  Biology, 
and  Allied  Sciences.  Bei«g  an  Exhaustive  Lexicon  of  Medicine 
and  those  Sciences  Collateral  to  it:  Biology  (Zoology  and  Botany), 
Chemistry,  Dentistry,  Parmacology,  Microscopy,  etc.,  with  many 
useful  Tables  and  numerous  fine  Illustrations.  1633  pages.  3d  Ed. 
Sheep  or  Half  Dark  Green  Leather,  ^10.00;  Thumb  Index,  $11.00 
Half  Russia,  Thumb  Index,  $12.00 
GOULD.  The  Medical  Student’s  Dictionary.  Including  all  the 
Words  and  Phrases  Generally  Used  in  Medicine,  with  their  Proper 
Pronunciation  and  Definition,  Based  on  Recent  Medical  Literature. 
With  Tables  of  the  Bacilli,  Micrococci,  Mineral  Springs,  etc.,  of  the 
Arteries,  Muscles,  Nerves,  Ganglia,  and  Plexuses,  etc.  loth  Edition. 
Rewritten  and  Enlarged.  Completely  reset  from  new  type  700  pp. 

Half  Dark  Leather,  $3.25  ; Half  Morocco,  Thumb  Index,  $4  00 
GOULD.  The  Pocket  Pronouncing  Medical  Lexicon.  (12,000 
Medical  Words  Pronounced  and  Defined.)  Containing  all  the  Words, 
their  Definition  and  Pronunciation,  that  the  Medical,  Dental,  or 
Pharmaceutical  Student  Generally  Comes  in  Contact  With;  also 
Elaborate  Tables  of  the  Arteries,  Muscles,  Nerves,  Bacilli,  etc.,  etc., 
a Dose  List  in  both  English  and  Metric  System,  etc..  Arranged  in  a 
Most  Convenient  Form  for  Reference  and  Memorizing. 

Full  Limp  Leather,  Gilt  Edges,  $1.00;  Thumb  Index,  $1.25 
60,000  Copies  of  Gould’s  Dictionaries  Have  Been  Sold. 

***  Sample  Pages  and  Illustrations  and  Descriptive  Circulars  of 
Gould’s  Dictionaries  sent  free  upon  application. 

HARRIS.  Dictionary  of  Dentistry.  Including  Definitions  of  Such 
Words  and  Phrases  of  the  Collateral  Sciences  as  Pertain  to  the  Art 
and  Practice  of  Dentistry.  5th  Edition.  Revised  and  Enlarged  by 
Ferdinand  J.  S.  Gorgas,  m.d.,  d.d.s.  Cloth,  $4.50;  Leather,  $5.50 
LONGLEY.  Pocket  Medical  Dictionary.  With  an  Appendix, 
containing  Poisons  and  their  Antidotes,  Abbreviations  used  in  Pre- 
scriptions, etc.  Cloth,  .75  ; Tucks  and  Pocket,  $1.00 

CLEVELAND.  Pocket  Medical  Dictionary.  33d  Edition.  Very 
small  pocket  size.  Cloth,  .50  ; Tucks  with  Pocket,  .75 

MAXWELL.  Terminologia  Medica  Polyglotta.  By  Dr. 
Theodore  Maxwell,  Assisted  by  Others.  $3.00 

The  object  of  this  work  is  to  assist  the  medical  men  of  any  nationality 
in  reading  medical  literature  written  in  a language  not  their  own. 
Each  term  is  usually  given  in  seven  languages,  viz. : English,  French, 
German,  Italian,  Spanish,  Russian,  and  Latin. 

TREVES  AND  LANG.  German-English  Medical  Dictionary. 

Half  Russia,  $3.25 

EAR  (see  also  Throat  and  Nose). 

HOVELL.  Diseases  of  the  Ear  and  Naso-Pharynx.  Includ- 
ing Anatomy  and  Physiology  of  the  Organ,  together  with  the  Treat- 
ment of  the  Affections  of  the  Nose  and  Pharynx  which  Conduce  to 
Aural  Disease.  122  Illustrations.  $5.00 

BURNETT.  Hearing  and  How  to  Keep  It.  Illustrated.  .40 
DALBY.  Diseases  and  Injuries  of  the  Ear.  4th  Edition.  38 
Wood  Engravings  and  8 Colored  Plates.  $2.50 

PRITCHARD.  Diseases  of  the  Ear.  3d  Edition,  Enlarged. 

Many  Illustrations  and  Formulae.  Just  Ready.  $1.50 

WOAKES.  Deafness,  Giddiness,  and  Noises  in  the  Head. 
4th  Edition.  Illustrated.  Just  Ready.  $2.00 


MEDICAL  BOOKS. 


9 


ELECTRICITY. 

BIGELOW.  Plain  Talks  on  Medical  Electricity  and  Bat- 
teries. With  a Therapeutic  Index  ^nd  a Glossary.  43  Illustra- 
tions. 2d  Edition.  $1.00 

JONES.  Medical  Electricity.  2d  Edition.  112  Illustrations.  $2.50 
MASON.  Electricity  ; Its  Medical  and  Surgical  Uses.  Numer- 
ous Illustrations.  .75 

EYE. 

A special  Circular  of  Books  on  the  Eye  sent  free  upon  application. 

ARLT.  Diseases  of  the  Eye.  Clinical  Studies  on  Diseases  of  the 
Eye.  Authorized  Translation  by  Lyman  Ware,  m.d.  Illustrated. 

fi.25 

PICK.  Diseases  of  the  Eye  and  Ophthalmoscopy.  Trans- 
lated by  A.  B.  Hale,  m d.  157  Illustrations,  many  of  which  are  in 
colors,  and  a glossary.  Just  Ready.  Cloth,  ^4.50  ; Sheep,  $5.50 
GOULD  AND  PYLE.  Compend  of  Diseases  of  the  Eye  and 
Refraction.  Including  Treatment  and  Operations,  and  a Section 
on  Local  Therapeutics.  With  Formulae,  Useful  Tables,  and  iii 
Illustrations,  several  of  which  are  in  colors.  Just  Ready. 

Cloth,  80;  Interleaved,  $1.00 
GOWERS.  Medical  Ophthalmoscopy.  A Manual  and  Atlas 
with  Colored  Autotype  and  Lithographic  Plates  and  Wood-cuts, 
Comprising  Original  Illustrations  of  the  Changes  of  the  Eye  in  Dis- 
eases of  the  Brain,  Kidney,  etc.  3d  Edition.  ^4.00 

HARLAN.  Eyesight,  and  How  to  Care  for  It.  Ulus.  .40 

HARTRIDGE.  Refraction.  96  Illustrations  and  Test  Types. 

8th  Edition,  Enlarged.  ^1.50 

HARTRIDGE.  On  the  Ophthalmoscope.  2d  Edition.  With 
Colored  Plate  and  many  Wood-cuts.  $1-25 

HANSELL  AND  BELL.  Clinical  Ophthalmology.  Colored 
Plate  of  Normal  Fundus  and  120  Illustrations.  $1.50 

MACNAMARA.  On  the  Eye.  5th  Edition.  Numerous  Colored 
Plates,  Diagrams  of  Eye,  Wood-cuts,  and  Test  Types.  $3-5o 

MORTON.  Refraction  of  the  Eye.  Its  Diagnosis  and  the  Cor- 
rection of  its  Errors.  With  Chapter  on  Keratoscopy  and  Test 
Types.  6th  Edition.  $1.00 

OHLEMANN.  Ocular  Therapeutics.  Authorized  Translation, 
and  Edited  by  Dr.  Charles  A.  Oliver.  In  Press. 

PHILLIPS.  Spectacles  and  Eyeglasses.  Their  Prescription 
and  Adjustment.  2d  Edition.  49  Illustrations.  ^i.oo 

SWANZY.  Diseases  of  the  Eye  and  Their  Treatment.  6th 
Edition,  Revised  and  Enlarged.  158  Illustrations,  i Plain  Plate, 
and  a Zephyr  Test  Card.  Just  Ready.  $3.00 

THORINGTON.  Retinoscopy.  Illustrated.  Just  Ready,  ^i.oo 
WALKER.  Students’  Aid  in  Ophthalmology.  Colored  Plate 
and  40  other  Illustrations  and  Glossary.  $1.50 

FEVERS. 

COLLIE.  On  Fevers.  Their  History,  Etiology,  Diagnosis,  Prog- 
nosis, and  Treatment.  Colored  Plates.  $2.00 

GOODALL  AND  WASHBOURN.  Fevers  and  Their  Treat- 
ment. Illustrated.  Just  Ready . $3.00 


10 


SUBJECT  CATALOGUE. 


GOUT  AND  RHEUMATISM. 

DUCKWORTH.  A Treatise  on  Gout.  With  Chromo-lithographs 
and  Engravings.  ^ Cloth,  $6.00 

GARROD.  On  Rheumatism.  A Treatise  on  Rheumatism  and 
Rheumatic  Arthritis,  Cloth,  $5.00 

HAIG.  Causation  of  Disease  by  Uric  Acid.  A Contribution  to 
the  Pathology  of  High  Arterial  Tension,  Headache,  Epilepsy,  Gout, 
Rheumatism,  Diabetes,  Bright's  Disease,  etc.  3d  Edition.  $3-oo 


HEADACHES. 

DAY.  On  Headaches.  The  Nature,  Causes,  and  Treatment  of 
Headaches.  4th  Edition  Illustrated.  $1.00 

HEALTH  AND  DOMESTIC  MEDI- 
CINE  (see  also  Hygiene  and  Nursing). 


BUCKLEY.  The  Skin  in  Health  and  Disease.  Ulus.  .40 

BURNETT.  Hearing  and  How  to  Keep  It.  Illustrated.  .40 
COHEN.  The  Throat  and  Voice.  Illustrated  .40 

DULLES.  Emergencies.  4th  Edition.  Illustrated.  ^i.oo 

HARLAN.  Eyesight  and  How  to  Care  for  It.  Illustrated.  .40 
HARTSHORNE.  Our  Homes.  Illustrated.  .40 

OSGOOD.  The  Winter  and  its  Dangers.  .40 

PACKARD.  Sea  Air  and  Bathing.  .40 

PARKES.  The  Elements  of  Health.  Just  Ready.  $1-25 

RICHARDSON.  Long  Life  and  How  to  Reach  It.  .40 

WESTLAND.  The  Wife  and  Mother.  $1.50 

WHITE.  The  Mouth  and  Teeth.  Illustrated.  .40 

WILSON.  The  Summer  and  its  Diseases.  .40 

WOOD.  Brain  Work  and  Overwork.  .40 

STARR.  Hygiene  of  the  Nursery.  5th  Edition.  $1.00 

CANFIELD.  Hygiene  of  the  Sick-Room.  ^125 


HEART. 

SANSOM.  Diseases  of  the  Heart.  The  Diagnosis  and  Pathology 
of  Diseases  of  the  Heart  and  Thoracic  Aorta.  With  Plates  and  other 
Illustrations.  $6.00 


HISTOLOGY. 

STIRLING.  Outlines  of  Practical  Histology.  368  Illustrations. 

2d  Edition,  Revised  and  Enlarged.  With  new  Illustrations.  J2.00 
STOHR.  Histology  and  Microscopical  Anatomy.  Translated 
and  Edited  by  A.  Shaper,  m.d.,  Harvard  Medical  School.  268 
Illustrations.  Just  Ready.  $3-oo 


MEDICAL  BOOKS. 


11 


HYGIENE  AND  WATER  ANALYSIS. 

special  Catalogue  of  Books  on  Hygiene  sent  free  upon  application. 

CANFIELD.  Hygiene  of  the  Sick-Room.  A Book  for  Nurses 
and  Others  Being  a Brief  Consideration  of  Asepsis,  Antisepsis,  Dis- 
infection, Bacteriology,  Immunity,  Heating  and  Ventilation,  and 
Kindred  Subjects.  $1.25 

COPLIN  AND  BEVAN.  Practical  Hygiene.  A Complete 
American  Text-Book.  138  Illustrations.  Cloth,  $3.25  ; Sheep,  ^4.25 
FOX.  Water,  Air,  and  Food.  Sanitaiy  Examinations  of  Water, 
Air,  and  Food.  100  Engravings.  2d  Edition,  Revised.  ^3*50 

KENWOOD.  Public  Health  Laboratory  Work.  116  Illustra- 
tions and  3 Plates.  ^2.00 

LEFFMANN.  Examination  of  Water  for  Sanitary  and 
Technical  Purposes.  3d  Edition.  Illustrated.  $1.25 

LEFFMANN.  Analysis  of  Milk  and  Milk  Products.  Illus- 
trated. $1.25 

LINCOLN.  School  and  Industrial  Hygiene.  .40 

MACDONALD.  Microscopical  Examinations  of  Water  and 
Air.  25  Lithographic  Plates,  Reference  Tables,  etc.  2d  Ed.  $2.50 
McNEILL.  The  Prevention  of  Epidemics  and  the  Construc- 
tion and  Management  of  Isolation  Hospitals.  Numerous  Plans 
and  Illustrations.  i^3-5o 

NOTTER  ANb  FIRTH.  The  Theory  and  Practice  of  Hygiene. 
(Being  the  9th  Edition  of  Parkes*  Practical  Hygiene,  rewritten  and 
brought  up  to  date.)  10  Plates  and  135  other  Illustrations.  1034 
pages.  8vo.  Just  Ready.  $7.00 

PARKES.  Hygiene  and  Public  Health.  By  Louis  C.  Parkes, 
M.D,  5th  Edition.  Enlarged.  Illustrated.  In  Press. 

PARKES.  Popular  Hygiene.  The  Elements  of  Health.  A Book 
for  Lay  Readers.  Illustrated. 

STARR.  The  Hygiene  of  the  Nursery.  Including  the  General 
Regimen  and  Feeding  of  Infants  and  Children,  and  the  Domestic 
Management  of  the  Ordinary  Emergencies  of  Early  Life,  Massage, 
etc.  5th  Edition.  25  Illustrations.  Just  Ready,  %\.oo 

STEVENSON  AND  MURPHY.  A Treatise  on  Hygiene.  By 
Various  Authors.  In  Three  Octave  Volumes.  Illustrated. 

Vol.  I,  $6.00;  Vol.  II,;^6.oo;  Vol.  Ill,  $5.00 
***  Each  Volume  sold  separately.  Special  Circular  upon  application. 

WILSON.  Hand-Book  of  Hygiene  and  Sanitary  Science. 

With  Illustrations.  7th  Edition.  $3.00 

WEYL.  Sanitary  Relations  of  the  Coal-Tar  Colors.  Author- 
ized Translation  by  Henry  Leffmann,  m.d.,  ph.d.  $1.25 

***  special  Catalogue  0/  Books  on  Hygiene  free  upon  application. 


JOURNALS,  ETC. 

OPHTHALMIC  REVIEW.  A Monthly  Record  of  Ophthalmic 
Science.  Publ.  in  London.  Sample  number  .25 ; per  annum  ^3.00 
NEW  SYDENHAM  SOCIETY  PUBLICATION.  Three  to  six 
volumes  each  year.  Circular  upon  application.  Per  annum  ^8.00 


12 


SUBJECT  CATALOGUE. 


KIDNEY  DISEASES. 

THORNTON.  The  Surgery  of  the  Kidney.  19  Ulus.  Clo.,  $1.50 
TYSON.  Bright’s  Disease  and  Diabetes.  With  Especial  Ref- 
erence to  Pathology  and  Therapeutics.  Including  a Section  on  Reti- 
nitis in  Bright’s  Disease.  New  Edition.  In  Preparation 


LUNGS  AND  PLEURAE. 

HARRIS  AND  BEALE.  Treatment  of  Pulmonary  Consump- 
tion. $2.50 

POWELL.  Diseases  of  the  Lungs  and  Pleurae,  including 

Consumption.  Colored  Plates  and  other  Ulus.  4th  Ed.  ^4.00 

TUSSEY,  High  Altitudes  in  the  Treatment  of  Consumption. 
fust  Ready.  $'-5o 


MASSAGE. 

KLEEN.  Hand-Book  of  Massage.  Authorized  translation  by 
Mussey  Hartwell,  m.d.,  ph.d.  With  an  Introduction  by  Dr.  S. 
Weir  Mitchell.  Illustrated  by  a series  of  Photographs  Made 
Especially  by  Dr.  Kleen  for  the  American  Edition.  $2.25 

MURRELL.  Massotherapeutics.  Massage  as  a Mode  of  Treat- 
ment. 5th  Edition.  ♦ $1.25 

OSTROM.  Massage  and  the  Original  Swedish  Move- 
ments. Their  Application  to  Various  Diseases  of  the  Body.  A 
Manual  for  Students,  Nurses,  and  Physicians.  Third  Edition,  En- 
larged. 94  Wood  Engravings,  many  of  which  are  original.  ^i.oo 


MATERIA  MEDICA  AND  THERA- 
PEUTICS. 

ALLEN,  HARLAN,  HARTE,  VAN  HARLINGEN.  A 
Hand-Book  of  Local  Therapeutics,  Beinga  Practical  Description 
of  all  those  Agents  Used  in  the  Local  Treatment  of  Diseases  of  the 
Eye,  Ear,  Nose  and  Throat,  Mouth,  Skin,  Vagina,  Rectum,  etc., 
such  as  Ointments,  Plasters,  Powders,  Lotions,  Inhalations,  Supposi- 
tories, Bougies,  Tampons,  and  the  Proper  Methods  of  Preparing  and 
Applying  Them.  Cloth,  $3.00  ; Sheep,  ^14. 00 

BIDDLE.  Materia  Medica  and  Therapeutics.  Including  Dose 
List,  Dietary  for  the  Sick,  Table  of  Parasites,  and  Memoranda  of 
New  Remedies.  13th  Edition,  Thoroughly  Revised  in  accord- 
ance with  the  new  U.  S.  P.  64  Illustrations  and  a Clinical  Index. 

Cloth,  $4.00;  Sheep,  1^5.00 
BRACKEN.  Outlines  of  Materia  Medica  and  Pharmacology.  By 
H.  M.  Bracken,  University  of  Minnesota.  ^2.75 

DAVIS.  Materia  Medica  and  Prescription  Writing.  $1.50 
FIELD.  Evacuant  Medication.  Cathartics  and  Emetics.  $1.75 
GORGAS.  Dental  Medicine.  A Manual  of  Materia  Medica  and 
Therapeutics  5th  Edition,  Revised.  $4.00 

HELLER.  Essentials  of  Materia  Medica,  Pharmacy,  and 
Prescription  Writing.  In  Press. 

MAYS.  Theine  in  the  Treatment  of  Neuralgia.  % bound,  .50 


MEDICAL  BOOKS 


13 


NAPHEYS.  Modern  Therapeutics,  gth  Revised  Edition,  En- 
larged and  Improved.  In  two  handsome  volumes.  Edited  by  Allen 
J.  Smith,  m.d.,  and  J.  Aubrey  Davis,  m.d. 

Vol.  I.  General  Medicine  and  Diseases  of  Children.  ^4.00 

Vol.  II.  General  Surgery,  Obstetrics,  and  Diseases  of  Women.  ^4.00 
POTTER.  Hand-Book  of  Materia  Medica,  Pharmacy,  and 
Therapeutics,  including  the  Action  of  Medicines,  Special  Therapeu- 
tics, Pharmacology,  etc.,  including  over  600  Prescriptions  and  For- 
mulae. 6th  Edition,  Revised  and  Enlarged.  With  Thumb  Index  in 
each  copy.  Cloth,  ^4.50;  Sheep,  $5.50 

POTTER.  Compend  of  Materia  Medica,  Therapeutics,  and 
Prescription  Writing,  with  Special  Reference  to  the  Physiologi- 
cal Action  of  Drugs.  6th  Revised  and  Improved  Edition,  based  upon 
the  U.  S.  P.  1890.  .80;  Interleaved,  $1.25 

SAYRE.  Organic  Materia  Medica  and  Pharmacognosy.  An 
Introduction  to  the  Study  of  the  Vegetable  Kingdom  and  the  Vege- 
table and  Animal  Drugs.  Comprising  the  Botanical  and  Physical 
Characteristics,  Source,  Constituents,  and  Pharmacopeial  Preparar 
tions.  With  chapters  on  Synthetic  Organic  Remedies,  Insects  In- 
jurious to  Drugs,  and  Pharmacal  Botany.  A Glossary  and  543  Illus- 
trations, many  of  which  are  original.  $4.00 

WARING.  Practical  Therapeutics.  4th  Edition,  Revised  and 
Rearranged.  Cloth,  ;^2.oo ; Leather,  $3.00 

WHITE  AND  WILCOX.  Materia  Medica,  Pharmacy,  Phar- 
macology, and  Therapeutics.  3d  American  Edition,  Revised  by 
Reynold  W.  Wilcox,  m.a.,  m.d.,  ll.d.  Clo.,  $2.75;  Lea.,  $3.25 


MEDICAL  JURISPRUDENCE  AND 
TOXICOLOGY. 

REESE.  Medical  Jurisprudence  and  Toxicology.  A Text-Book 
for  Medical  and  Legal  Practitioners  and  Students.  4th  Edition. 
Revised  by  Henry  Leffmann,  m.d.  Clo. ,^3.00;  Leather,  $3.50 

“ To  the  student  of  medical  jurisprudence  and  toxicology  it  is  in- 
valuable, as  it  is  concise,  clear,  and  thorough  in  every  respect.” — The 
American  Journal  of  the  Medical  Sciences. 

MANN.  Forensic  Medicine  and  Toxicology.  Ulus.  1^6.50 
MURRELL.  What  to  Do  in  Cases  of  Poisoning.  7th 
Edition,  Enlarged.  |$i.oo 

TANNER.  Memoranda  of  Poisons.  Their  Antidotes  and  Tests. 
7th  Edition.  .75 

MICROSCOPY. 

BEALE.  The  Use  of  the  Microscope  in  Practical  Medicine. 
For  Students  and  Practitioners,with  Full  Directions  for  Examining  the 
Various  Secretions, etc.,  by  the  Microscope.  4th  Ed.  500  Ulus.  ^6.50 
BEALE.  How  to  Work  with  the  Microscope.  A Complete 
Manual  of  Microscopical  Manipulation,  containing  a Full  Description 
of  many  New  Processes  of  Investigation,  with  Directions  for  Examin- 
ing Objects  Under  the  Highest  Powers,  and  for  Taking  Photographs 
of  Microscopic  Objects.  5th  Edition.  400  Illustrations,  many  of 
them  colored.  $6.50 

CARPENTER.  The  Microscope  and  Its  Revelations.  7th 
Edition.  800  Illustrations  and  many  Lithographs.  $5- 50 


14 


SUBJECT  CATALOGUE. 


LEE.  The  Microtomist’s  Vade  Mecum.  A Hand-Book  of 
Methods  of  Microscopical  Anatomy.  887  Articles.  4th  Edition, 
Enlarged.  Just  Ready.  $4.00 

MACDONALD.  Microscopical  Examinations  of  Water  and  Air. 

25  Lithographic  Plates,  Reference  Tables,  etc.  2d  Edition.  $2.50 
REEVES.  Medical  Microscopy,  including  Chapters  on  Bacteri- 
ology, Neoplasms,  Urinary  Examination,  etc.  Numerous  Illus- 
trations, some  of  which  are  printed  in  colors.  , $2.50 

WETHERED.  Medical  Microscopy.  A Guide  to  the  Use  of  the 
Microscope  in  Practical  Medicine.  100  Illustrations.  $2.00 

MISCELLANEOUS. 

BLACK.  Micro-Organisms.  The  Formation  of  Poisons.  A 
Biological  Study  of  the  Germ  Theory  of  Disease.  .75 

BURNETT.  Foods  and  Dietaries.  A Manual  of  Clinical  Diet- 
etics. 2d  Edition.  $1.50 

GOULD.  Borderland  Studies.  Miscellaneous  Addresses  and 
Essays.  i2mo.  ^2.00 

GOWERS.  The  Dynamics  of  Life.  .75 

HAIG.  Causation  of  Disease  by  Uric  Acid.  A Contribution  to 
the  Pathology  of  High  Arterial  Tension,  Headache,  Epilepsy,  Gout, 
Rheumatism,  Diabetes,  Bright's  Disease,  etc.  3d  Edition.  $3.00 
HARE.  Mediastinal  Disease.  ' Illustrated  by  six  Plates.  $2.00 
HEMMETER.  Diseases  of  the  Stomach.  Ulus.  In  Press, 
HENRY.  A Practical  Treatise  on  Anemia.  Half  Cloth,  .50 
LEFFMANN.  The  Coal-Tar  Colors.  With  Special  Reference  to 
their  Injurious  Qualities  and  the  Restrictions  of  their  Use.  A Trans- 
lation of  Theodore  Weyl's  Monograph.  $1.25 

TREVES.  Physical  Education  : Its  Effects,  Methods,  Etc.  .75 
LIZARS.  The  Use  and  Abuse  of  Tobacco.  .40 

PARRISH.  Alcoholic  Inebriety  from  a Medical  Standpoint, 
with  Cases.  $1.00 

ST.  CLAIR.  Medical  Latin.  $1.00 


BEEVOR. 

ment. 


NERVOUS  DISEASES. 

Diseases  of  the  Nervous  System  and  their  Treat- 

In  Press. 


GOWERS.  Manual  of  Diseases  of  the  Nervous  System.  A 

Complete  Text-Book.  2d  Edition,  Revised,  Enlarged,  and  in  many 
parts  Rewritten.  With  many  new  Illustrations.  Two  volumes. 

Vol.  I.  Diseases  of  the  Nerves  and  Spinal  Cord.  Clo.  $3.00  ; Sh.  $4.00 
Vol.  II.  Diseases  of  the  Brain  and  Cranial  Nerves;  General  and 
Functional  Disease.  Cloih,  $4.00 ; Sheep,  $5.00 


GOWERS.  Syphilis  and  the  Nervous  System.  $1.00 

GOWERS.  Diagnosis  of  Diseases  of  the  Brain.  2d  Edition. 
Illustrated. 

GOWERS.  Clinical  Lectures.  A New  Volume  of  Essays  on  the 
Diagnosis,  Treatment,  etc.,  of  Diseases  of  the  Nervous  System.  $2.00 


GOWERS.  Epilepsy  and  Other  Chronic  Convulsive  Diseases. 

2d  Edition.  In  Press. 

HORSLEY.  The  Brain  and  Spinal  Cord.  The  Structure  and 
Functions  of.  Numerous  Illustrations.  $2.50 


MEDICAL  BOOKS. 


15 


OBERSTEINER,  The  Anatomy  of  the  Central  Nervous  Or- 
gans. A Guide  to  the  Study  of  their  Structure  in  Health  and  Dis- 
ease. 198  Illustrations.  $5*5o 

ORMEROD.  Diseases  of  the  Nervous  System.  66  Wood  En- 
gravings. ^i.oo 

OSLER.  Cerebral  Palsies  of  Children.  A Clinical  Study.  $2.00 
OSLER.  Chorea  and  Choreiform  Affections.  $2.00 

PAGE.  Railroad  Injuries.  With  Special  Reference  to  Those  of  the 
Back  and  Nervous  System.  ^2.25 

PRESTON.  Hysteria  and  Certain  Allied  Conditions.  Their 
Nature  and  Treatment.  Illustrated.  Just  Ready.  $2.00 

THORBURN.  Surgery  of  the  Spinal  Cord.  Illustrated.  $4.00 
WATSON.  Concussions.  An  Experimental  Study  of  Lesions  Aris- 
ing from  Severe  Concussions,  Paper  cover^  $1.00 

WOOD.  Brain  Work  and  Overwork.  .40 

NURSING. 

special  Catalogue  of  Books  for  Nurses  sent  free  upon  application. 
BROWN.  Elementary  Physiology  for  Nurses.  .75 

CANFIELD.  Hygiene  of  the  Sick-Room.  A Book  for  Nurses  and 
Others.  Being  a Brief  Consideration  of  Asepsis,  Antisepsis,  Disinfec- 
tion, Bacteriology,  Immunity,  Heating  and  Ventilation,  and  Kindred 
Subjects  for  the  Use  of  Nurses  and  Other  Intelligent  Women.  $1.25 
CULLINGWORTH.  A Manual  of  Nursing,  Medical  and  Sur- 
gical. 3d  Edition  with  Illustrations.  . .75 

CULLINGWORTH.  A Manual  for  Monthly  Nurses.  3d  Ed.  .40 
CUFF,  Lectures  to  Nurses  on  Medicine.  25  Illustrations.  $1.00 
DOMVILLE.  Manual  for  Nurses  and  Others  Engaged  in  At- 
tending the  Sick.  8th  Edition.  With  Recipes  for  Sick-room  Cook- 
ery, etc.  .75 

FULLERTON,  Obstetric  Nursing.  40  Ills.  4th  Ed.  $1.00 
FULLERTON.  Nursing  in  Abdominal  Surgery  and  Diseases 
of  Women.  Comprising  the  Regular  Course  of  Instruction  at  the 
Training-School  of  the  Women’s  Hospital,  Philadelphia.  2d  Edition. 
70  Illustrations.  $1.50 

HUMPHREY.  A Manual  for  Nurses.  Including  General 
Anatomy  and  Physiology,  Management  of  the  Sick-Room,  etc.  15th 
Edition.  Illustrated.  ^i.oo 

SHAWE.  Notes  for  Visiting  Nurses,  and  all  those  Interested 
in  the  Working  and  Organization  of  District,  Visiting,  or 
Parochial  Nurse  Societies.  With  an  Appendix  Explaining  the 
Organization  and  Working  of  Various  Visiting  and  District  Nurse  So- 
cieties, by  Helen  C.  Jenks,  of  Philadelphia.  $1.00 

STARR.  The  Hygiene  of  the  Nursery.  Including  the  General 
Regimen  and  Feeding  of  Infants  and  Children,  and  the  Domestic  Man- 
agement of  the  Ordinary  Emergencies  of  Early  Life,  Massage,  etc.  5th 
Edition.  25  Illustrations.  Just  Ready.  ^i.oo 

TEMPERATURE  CHARTS.  For  Recording  Temperature,  Res- 
piration, Pulse,  Day  of  Disease,  Date,  Age,  Sex,  Occupation, 
Name,  etc.  Put  up  in  pads  of  fifty.  Each  .50 

VOSWINKEL.  Surgical  Nursing,  in  Illustrations.  ^i.oo 

***  special  Catalogue  of  Books  on  Nursing  free  upon  application. 


16 


SUBJECT  CATALOGUE. 


OBSTETRICS. 

BAR.  Antiseptic  Midwifery.  The  Principles  of  Antiseptic  Meth- 
ods Applied  to  Obstetric  Practice.  Authorized  Translation  by 
Henry  D.  Fry,  m.d.  , with  an  Appendix  by  the  Author.  $i.oo 

CAZJEAUX  AND  TARNIER.  Midwifery.  With  Appendix  by 
Mund6.  The  Theory  and  Practice  of  Obstetrics,  including  the  Dis- 
eases of  Pregnancy  and  Parturition,  Obstetrical  Operations,  etc. 
8th  Edition.  Illustrated  by  Chromo- Lithographs,  Lithographs,  and 
other  full-page  Plates,  seven  of  which  are  beautifully  colored,  and 
numerous  Wood  Engravings.  Cloth,  1^4.50  ; Full  Leather,  ^5.50 

DAVIS.  A Manual  of  Obstetrics.  Being  a Complete  Manual  for 
Physicians  and  Students.  2d  Edition.  16  Colored  and  other  Plates 
and  134  other  Illustrations.  ^2.00 

JELLETT.  The  Practice  of  Midwifery.  Illustrated.  In  Press. 
LANDIS.  Compend  of  Obstetrics.  5th  Edition,  Revised  by  Wm. 
H.  Wells,  Assistant  Demonstrator  of  Clinical  Obstetrics,  Jefferson 
Medical  College.  With  many  Illustrations,  .80  ; Interleaved,  ^1.25. 
SCHULTZE.  Obstetrical  Diagrams.  Being  a series  of  20  Col- 
ored Lithograph  Charts,  Imperial  Map  Size,  of  Pregnancy  and  Mid- 
wifery, with  accompanying  explanatory  (German)  text  illustrated 
by  Wood  Cuts.  2d  Revised  Edition. 

Price  in  Sheets,  1^26.00  ; Mounted  on  Rollers,  Muslin  Backs,  $36,00 
STRAHAN.  Extra-Uterine  Pregnancy.  The  Diagnosis  and 
Treatment  of  Extra-Uterine  Pregnancy.  .75 

\VINCKEL.  Text-Book  of  Obstetrics,  Including  the  Pathol- 
ogy and  Therapeutics  of  the  Puerperal  State.  Authorized 
Translation  by  J.  Clifton  Edgar,  a.m.,  m.d.  With  nearly  200  Illus- 
trations. Cloth,  $5.00 ; Leather,  $6.00 

FULLERTON.  Obstetric  Nursing.  4th  Ed.  Illustrated.  $1.00 
SHIBATA.  Obstetrical  Pocket-Phantom  with  Movable  Child 
and  Pelvis.  Letter  Press  and  Illustrations.  $1.00 


PATHOLOGY. 

BLACKBURN.  Autopsies.  A Manual  of  Autopsies  Designed  for 
the  Use  of  Hospitals  for  the  Insane  and  other  Public  Institutions. 
Ten  full-page  Plates  and  other  Illustrations.  $1.25 

BLODGETT.  Dental  Pathology.  By  Albert  N.  Blodgett, 
M.D.,  late  Professor  of  Pathology  and  Therapeutics,  Boston  Dental 
College.  33  Illustrations.  $1.25 

GILLIAM.  Pathology.  A Hand-Book  for  Students.  47  Ulus.  .75 
HALL.  Compend  of  General  Pathology  and  Morbid  Anatomy. 

91  very  fine  Illustrations.  .80;  Interleaved,  $1.25 

VIRCHOW.  Post-Mortem  Examinations.  A Description  and 
Explanation  of  the  Method  of  Performing  Them  in  the  Dead  House 
of  the  Berlin  Charity  Hospital,  with  Special  Reference  to  Medico- 
Legal  Practice.  3d  Edition,  with  Additions.  .75 


PHARMACY. 

special  Catalogue  0/  Books  on  Pharmacy  sent  free  upon  application. 

COBLENTZ.  Manual  of  Pharmacy.  A New  and  Complete 
Text-Book  by  the  Professor  in  the  New  York  College  of  Pharmacy. 
2d  Edition,  Revised  and  Enlarged.  437  Ulus.  Cloth,  $3.50 ; Sh.,  $4  50 


MEDICAL  BOOKS. 


17 


BEASLEY.  Book  of  3100  Prescriptions.  Collected  from  the 
Practice  of  the  Most  Eminent  Physicians  and  Surgeons — English, 
French,  and  American.  A Compendious  History  of  the  Materia 
Medica,  Lists  of  the  Doses  of  all  the  Officinal  and  Established  Pre- 
parations, an  Index  of  Diseases  and  their  Remedies.  7th  Ed.  $2.00 
BEASLEY.  Druggists’  General  Receipt  Book.  Comprising 
a Copious  Veterinary  Formulary,  Recipes  in  Patent  and  Proprietary 
Medicines,  Druggists’  Nostrums,  etc. ; Perfumery  and  Cosmetics, 
Beverages,  Dietetic  Articles  and  Condiments,  Trade  Chemicals, 
Scientific  Processes,  and  an  Appendix  of  Useful  Tables.  loth  Edi- 
tion, Revised.  ;^2.oo 

BEASLEY.  Pocket  Formulary.  A Synopsis  of  the  British  and 
Foreign  Pharmacopoeias,  Comprising  Standard  and  Approved 
Formulae  for  the  Preparations  and  Compounds  Employed  in  Medical 
Practice,  nth  Edition.  ^2  00 

PROCTOR.  Practical  Pharmacy.  Lectures  on  Practical  Phar- 
macy. With  Wood  Engravings  and  32  Lithographic  Fac-simile 
Prescriptions.  3d  Edition,  Revised,  and  with  Elaborate  Tables  of 
Chemical  Solubilities,  etc,  $3-oo 

ROBINSON.  Latin  Grammar  of  Pharmacy  and  Medicine. 

2d  Edition.  With  elaborate  Vocabularies.  $^  75 

SAYRE.  Organic  Materia  Medica  and  Pharmacognosy.  An 
Introduction  to  the  Study  of  the  Vegetable  Kinedom  and  the  Vege- 
table and  Animal  Drugs,  Comprising  the  Botanical  and  Physical 
Characteristics,  Source,  Constituents,  and  Pharmacopeial  Prepar- 
ations. With  Chapters  on  Synthetic  Organic  Remedies,  Insects 
Injurious  to  Drugs,  and  Pharmacal  Botany.  A Glossary  and  543 
Illustrations,  many  of  which  are  original.  Cloth,  ^4.00;  Sheep,  ^5.00 
SCOVILLE.  The  Art  of  Compounding.  A Text-Book  for  the 
Student  and  a Reference  Book  for  the  Pharmacist.  Cl. $2. 50;  Sh.^3.50 
STEWART.  Compend  of  Pharmacy.  Based  upon  **  Reming- 
ton’s l ext-Book  of  Pharmacy.”  5th  Edition,  Revised  in  Accord- 
ance with  the  U.  S.  Pharmacopoeia,  1890.  Complete  Tables  of 
Metric  and  English  Weights  and  Measures.  .80;  Interleaved,  $1,25 
UNITED  STATES  PHARMACOPCEIA.  1890.  7th  Decennial 
Revision.  Cloth,  $2.50  (postpaid,  $2.77);  Sheep,  $3.00  (postpaid, 
$3,27);  Interleaved,  ;^4. 00  (postpaid,  $4.50);  Printed  on  one  side  ot 
page  only,  unbound,  ^3.50  (postpaid,  553.90). 

Select  Tables  from  the  U.  S.  P.  (1890).  Being  Nine  of  the  Most 
Important  and  Useful  Tables,  Printed  on  Separate  Sheets.  Care- 
fully put  up  in  patent  envelope.  .25 

POTTER.  Hand-Book  of  Materia  Medica,  Pharmacy,  and 
Therapeutics.  600  Prescriptions  and  Formulae.  6th  Edition. 

Cloth,  $4  50;  Sheep,  $5.50 

***  Special  Catalogue  0/  Books  on  Pharmacy  free  ufon  application. 


PHYSICAL  DIAGNOSIS. 

FENWICK.  Medical  Diagnosis.  8th  Edition,  very  much  En- 
larged. Nearly  Ready. 

TYSON.  Hand-Book  of  Physical  Diagnosis.  For  Students  and 
Physicians.  By  the  Professor  of  Clinical  Medicine  in  the  University 
of  Pennsylvania.  Ulus.  2d  Ed.,  Improved  and  Enlarged.  $1-25 

MEMMINGER.  Diagnosis  by  the  Urine.  23  Ulus.  ^i.oo 

2 


18 


SUBJECT  CATALOGUE. 


PHYSIOLOGY. 

BRUBAKER.  Compend  of  Physiology.  8th  Edition,  Revised 
and  Enlarged.  Illustrated.  .80;  Interleaved,  $1.25 

KIRKE.  Physiology.  (14th  Authorized  Edition.  Dark-Red  Cloth.) 
A Hand-Book  of  Physiology.  14th  Edition,  Revised  and  Enlarged. 
By  Prof.  W.  D Hai.liburton,  of  Kings  College,  London.  661 
Illustrations,  some  of  which  are  printed  in  colors.  Just  Ready, 

Cloth,  $3.25;  Leather,  ^4.00 
LANDOIS.  A Text-Book  of  Human  Physiology,  Including 
Histology  and  Microscopical  Anatomy,  with  Special  Reference  to 
the  Requirements  of  Practical  Medicine.  5th  American,  translated 
from  the  9th  German  Edition,  with  Additions  by  Wm.  Stirling, 
M.D.,D.sc.  845  Ulus.,  many  of  which  are  printed  in  colors.  In  Press. 
STARLING.  Elements  of  Human  Physiology.  100 Ills,  ^i.oo 
STIRLING.  Outlines  of  Practical  Physiology.  Including 
Chemical  and  Experimental  Physiology,  with  Special  Reference  to 
Practical  Medicine.  3d  Edition.  289  Illustrations.  ^2.00 

TYSON.  Cell  Doctrine.  Its  History  and  Present  State.  $1.50 
YEO.  Manual  of  Physiology.  A Text- Book  for  Students  of 
Medicine.  By  Gerald  F.  Yeo,  m.d.,  f.r.c.s.  6th  Edition.  254 
Illustrations  and  a Glossary.  Cloth,  $2.50  ; Leather,  $3.00 

PRACTICE. 

BEALE.  On  Slight  Ailments;  their  Nature  and  Treatment. 

2d  Edition,  Enlarged  and  Illustrated.  ^1.25 

CHARTERIS.  Practice  of  Medicine.  6th  Edition.  $2.00 

FOWLER.  Dictionary  of  Practical  Medicine.  By  various 
writers.  An  Encyclopaedia  of  Medicine.  Clo.,^3.00;  Half  Mor.  $4.00 
HUGHES.  Compend  of  the  Practice  of  Medicine.  5th  Edition, 
Revised  and  Enlarged. 

Part  I.  Continued,  Eruptive,  and  Periodical  Fevers,  Diseases  of  the 
Stomach,  Intestines,  Peritoneum,  Biliary  Passages,  Liver,  Kid- 
neys, etc.,  and  General  Diseases,  etc. 

Part  II.  Diseases  of  the  Respiratory  System,  Circulatory  System, 
and  Nervous  System;  Diseases  of  the  Blood,  etc. 

Price  of  each  part,  .80;  Interleaved,  ^1.25 
Physician’s  Edition.  In  one  volume,  including  the  above  two 
parts,  a Section  on  Skin  Diseases,  and  an  Index.  5th  Revised, 
Enlarged  Edition.  568  pp.  Full  Morocco,  Gilt  Edge,  $2.25 

ROBERTS.  The  Theory  and  Practice  of  Medicine.  The 
Sections  on  Treatment  are  especially  exhaustive.  9th  Edition, 
with  Illustrations.  Cloth,  ^4.50;  Leather,  ^5,50 

TAYLOR.  Practice  of  Medicine.  Cloth,  ;^2.oo ; Sheep,  $2.50 
TYSON.  The  Practice  of  Medicine.  By  James  Tyson,  m.d.. 
Professor  of  Clinical  Medicine  in  the  University  of  Pennsylvania. 
A Complete  Systematic  Text-book  with  Special  Reference  to  Diag- 
nosis and  Treatment.  Illustrated.  8vo.  Just  Ready. 

Cloth,  ^5.50  ; Leather,  $6  50  ; Half  Russia,  ^7.50 

PRESCRIPTION  BOOKS. 

BEASLEY.  Book  of  3100  Prescriptions.  Collected  from  the 
Practice  of  the  Most  Eminent  Physicians  and  Surgeons — English, 
French,  and  American.  A Compendious  History  of  the  Materia, 
Medica,  Lists  of  the  Doses  of  all  Officinal  and  Established  Prepara- 
tions, and  an  Index  of  Diseases  and  their  Remedies.  7th  Ed.  ^2.00 


MEDICAL  BOOKS. 


19 


BEASLEY.  Druggists’  General  Receipt  Book.  Comprising 
a Copious  Veterinary  Formulary,  Recipes  m Patent  and  Proprie- 
tary Medicines,  Druggists’  Nostrums,  etc.  ; Perfumery  and  Cos- 
metics, Beverages,  Dietetic  Articles  and  Condiments,  Trade  Chem- 
icals, Scientific  Processes,  and  an  Appendix  of  Useful  Tables, 
loth  Edition,  Revised.  ^2.00 

BEASLEY.  Pocket  Formulary.  A Synopsis  of  the  British  and 
Foreign  Pharmacopoeias.  Comprising  Standard  and  Approved 
Formulae  for  the  Preparations  and  Compounds  Employed  in  Medical 
Practice,  iith  Edition.  Cloth,  $2.00 

PEREIRA.  Prescription  Book.  Containing  Lists  of  Phrases 
and  Abbreviations  Used  in  Prescriptions,  Grammatical  Construction 
of  Prescriptions,  etc.  i6th  Edition.  Cloth,  .75  ; Tucks,  $1.00 

WYTHE.  Dose  and  Symptom  Book.  Containing  the  Doses  and 
Uses  of  all  the  Principal  Articles  of  the  Materia  Medica.  17th  Ed. 

Cloth,  .75  ; Leather,  with  Tucks  and  Pocket,  ;Ji.oo 

SKIN. 

BULKLEY.  The  Skin  in  Health  and  Disease.  Illustrated.  .40 
CROCKER.  Diseases  of  the  Skin.  Their  Description,  Pathol- 
ogy, Diagnosis,  and  Treatment,  with  Special  Reference  to  the  Skin 
Eruptions  of  Children.  92  Ulus.  2d  Edition.  Cloth,  ^4.50 ; Sh.,  ^5.50 
IMPEY.  Leprosy.  37  Plates.  8vo.  $3-5o 

SCHAMBERG.  Diseases  of  the  Skin.  Illustrated.  Being  No. 

16  ? Quiz-Compend  ? Series.  Cloth,  .80;  Interleaved,  $1.25 

VAN  HARLINGEN.  On  Skin  Diseases.  A Practical  Manual 
of  Diagnosis  and  Treatment,  with  special  reference  to  Differential 
Diagnosis.  3d  Edition,  Revised  and  Enlarged.  With  Formulae 
and  60  Illustrations,  some  of  which  are  printed  in  colors.  $2.75 

SURGERY  AND  SURGICAL  DIS- 
EASES. 

CAIRD  AND  CATHCART.  Surgical  Hand-Book.  5th  Edition, 
Revised.  188  Illustrations.  Full  Red  Morocco,  ^2.50 

DEAVER.  Appendicitis,  Its  Symptoms,  Diagnosis,  Pathol- 
ogy, Treatment,  and  Complications.  Elaborately  Illustrated 
with  Colored  Plates  and  other  Illustrations.  Cloth,  $3.50 

DEAVER.  Surgical  Anatomy.  With  200  Illustrations,  Drawn  by  a 
Special  Artist  from  Directions  made  for  the  Purpose.  In  Preparation. 
DULLES.  What  to  Do  First  in  Accidents  and  Poisoning. 

4th  Edition.  New  Illustrations.  $1.00 

HACKER.  Antiseptic  Treatment  of  Wounds,  Introduction  to 
the.  According  to  the  Method  in  Use  at  Professor  Billroth’s  Clinic, 
Vienna.  With  a Photo-engraving  of  Billroth  in  his  Clinic.  .50 

HEATH.  Minor  Surgery  and  Bandaging.  loth  Ed  Revised 
and  Enlarged.  158  Illustrations,  62  Formulae,  Diet  List,  etc  ^1.25 
HEATH.  Injuries  and  Diseases  of  the  Jaws.  4th  Edition. 

187  Illustrations.  $4-5o 

HEATH.  Lectures  on  Certain  Diseases  of  the  Jaws.  64  Illus- 
trations. Boards,  .50 

HORWITZ.  Compend  of  Surgery  and  Bandaging,  including 
Minor  Surgery,  Amputations,  Fractures,  Dislocations,  Surgical  Dis- 
eases, and  the  Latest  Antiseptic  Rules,  etc.,  with  Differential  Diagno- 
sis and  Treatment.  5th  Edition,  very  much  Enlarged  and  Rear- 
ranged. 167  Illustrations,  98  Formulae.  Clo.,  .80;  Interleaved,  $125. 


20 


SUBJECT  CATALOGUE. 


JACOBSON.  Operations  of  Surgery.  Over  200  Illustrations. 

Cloth,  $3.00 ; Leather,  $4.00 
JACOBSON.  Diseases  of  the  Male  Organs  of  Generation. 

88  Illustrations,  $6.00 

MACREADY.  A Treatise  on  Ruptures.  24  Full-page  Litho- 
graphed Plates  and  Numerous  Wood  Engravings.  Cloth,  ^6.00 

MAYLARD.  Surgery  of  the  Alimentary  Canal.  134  Ulus.  $7.50 
MOULLIN.  Text-Book  of  Surgery.  With  Special  Reference  to 
Treatment.  3d  American  Edition.  Revised  and  edited  by  John  B. 
Hamilton,  m.d.,  ll.d..  Professor  of  the  Principles  of  Surgery  and 
Clinical  Surgery,  Rush  Medical  College,  Chicago.  623  Illustrations, 
over  200  of  which  are  original,  and  many  of  which  are  printed  in 
colors.  Handsome  Cloth,  ^6.00;  Leather,  7. 00 

‘*The  aim  to  make  this  valuable  treatise  practical  by  giving  special 
attention  to  questions  of  treatment  has  been  admirably  carried  out. 
Many  a reader  will  consult  the  work  with  a feeling  of  satisfaction  that 
his  wants  have  been  understood,  and  that  they  have  been  intelligently 
met.” — The  American  Journal  of  Medical  Science. 

SMITH.  Abdominal  Surgery.  Being  a Systematic  Description  of 
all  the  Principal  Operations.  224  Ulus.  5th  Ed.  2 Vols.  Clo.,  $10.00 
SWAIN.  Surgical  Emergencies.  Fifth  Edition.  . Cloth,  $1,75 
VOSWINKEL.  Surgical  Nursing,  in  Illustrations.  $1.00 
WALSHAM.  Manual  of  Practical  Surgery.  5th  Ed.,  Re- 
vised and  Enlarged.  With  380  Engravings.  Clo.,  $2.75  ; Lea.,  $3.25 
WATSON.  On  Amputations  of  the  Extremities  and  Their 
Complications.  250  Illustrations.  $5-5o 


THROAT  AND  NOSE  (see  also  Ear)* 

COHEN.  The  Throat  and  Voice.  Illustrated.  .40 

HALL.  Diseases  of  the  Nose  and  Throat.  Two  Colored 
Plates  and  59  Illustrations.  $2.50 

HUTCHINSON.  The  Nose  and  Throat.  Including  the  Nose, 
Naso- Pharynx,  Pharynx,  and  Larynx.  Illustrated  by  Lithograph 
Plates  and  40  other  Illustrations.  2d  Edition.  In  Press. 

MACKENZIE.  The  Pharmacopoeia  of  the  London  Hospital 
for  Diseases  of  the  Throat.  5th  Edition,  Revised  by  Dr.  F. 
G.  Harvey.  $1.00 

McBRIDE.  Diseases  of  the  Throat,  Nose,  and  Ear.  A Clinical 
Manual.  With  colored  Ulus,  from  original  drawings.  2d  Ed.  $6.00 
MURRELL.  Chronic  Bronchitis  and  its  Treatment.  (Author- 
ized Edition.)  A Clinical  Study.  $i-50 

POTTER.  Speech  and  its  Defects.  Considered  Physiologically, 

Pathologically,  and  Remedially.  $1.00 

WOAKES.  Post-Nasal  Catarrh  and  Diseases  of  the  Nose 

Causing  Deafness.  26  Illustrations.  $1.00 


URINE  AND  URINARY  ORGANS. 

ACTON.  The  Functions  and  Disorders  of  the  Reproductive 
Organs  in  Childhood,  Youth,  Adult  Age,  and  Advanced  Life, 
Considered  in  their  Physiological,  Social,  and  Moral  Relations. 
8th  Edition.  $i-75 


MEDICAL  BOOKS. 


21 


ALLEN.  Albuminous  and  Diabetic  Urine.  Ulus.  $2.25 

BROCKBANK.  Gallstones.  Just  Ready.  $2.25 

BEALE.  One  Hundred  Urinary  Deposits.  On  eight  sheets, 
for  the  Hospital,  Laboratory,  or  Surgery.  Paper,  ^2.00 

HOLLAND.  The  Urine,  the  Gastric  Contents,  the  Common 
Poisons,  and  the  Milk.  Memoranda,  Chemical  and  Microscopi- 
cal, for  Laboratory  Use.  Illustrated  and  Interleaved,  5th  Ed.  $1.00 
LEGG.  On  the  Urine.  7th  Edition,  Enlarged.  Ulus.  $1.00 

MEMMINGER.  Diagnosis  by  the  Urine.  23  Ulus.  ^i.oo 

MOULLIN.  Enlargement  of  the  Prostate.  Its  Treatment  and 
Radical  Cure,  Illustrated.  $150 

THOMPSON.  Diseases  of  the  Urinary  Organs.  8th  Ed.  $3.00 
TYSON.  Guide  to  Examination  of  the  Urine.  For  the  Use  of 
Physicians  and  Students.  With  Colored  Plate  and  Numerous  Illus- 
trations engraved  on  wood.  9th  Edition,  Revised.  $1*25 

VAN  NUYS.  Chemical  Analysis  of  Healthy  and  Diseased 
Urine,  Qualitative  and  Quantitative.  39  Illustrations.  |i.oo 


VENEREAL  DISEASES. 

COOPER.  Syphilis.  2d  Edition,  Enlarged  and  Illustrated  with 
20  full-page  Plates,  $5.00 

GOWERS.  Syphilis  and  the  Nervous  System.  100 

JACOBSON.  Diseases  of  the  Male  Organs  of  Generation.  88 

Illustrations.  ^6.00 


VETERINARY. 

ARMATAGE.  The  Veterinarian’s  Pocket  Remembrancer. 
Being  Concise  Directions  for  the  Treatment  of  Urgent  or  Rare  Cases, 
Embracing  Semeiology,  Diagnosis,  Prognosis,  Surgery,  Treatment, 
etc,  2d  Edition.  Boards,  $1.00 

BALLOU.  Veterinary  Anatomy  and  Physiology.  29  Graphic 
Illustrations.  .80;  Interleaved,  $1.25 

TUSON.  Veterinary  Pharmacopoeia.  Including  the  Outlines  of 
Materia  Medica  and  Therapeutics.  5th  Edition.  $2.25 


WOMEN,  DISEASES  OF. 

BYFORD  (H.  T.).  Manual  of  Gynecology.  With  234  Illustra- 
tions, many  of  which  are  from  original  drawings.  ^2  50 

BYFORD  (W.  H.).  Diseases  of  Women.  4th  Edition.  306 
Illustrations.  Cloth,  $2.00 

DUHRSSEN.  A Manual  of  Gynecological  Practice.  105 
Illustrations.  $150 

LEWERS.  Diseases  of  Women.  146  Ulus.  3d  Edition.  $2.00 
WELLS.  Compend  of  Gynecology.  Ulus.  .80;  Interleaved,  ^1.25 
WINCKEL.  Diseases  of  Women.  Translated  by  special  authority 
of  Author,  under  the  Supervision  of,  and  with  an  Introduction  by, 
Theophilus  Parvin,  m.d.  152  Engravings  on  Wood.  3d  Edition, 
Revised,  In  Preparation. 

FULLERTON.  Nursing  in  Abdominal  Surgery  and  Diseases 
of  Women.  2d  Edition.  70  Illustrations. 


22 


SUBJECT  CATALOGUE. 


COMPENDS. 


From  The  Southern  Clinic. 

**  We  know  of  no  series  of  books  issued  by  any  house  that  so  fully 
meets  our  approval  as  these  ? Quiz-Compends?.  They  are  well  ar- 
ranged, full,  and  concise,  and  are  really  the  best  line  of  text-books  that 
could  be  found  for  either  student  or  practitioner.'* 


BLAKISTON^S  ? QUIZ-COMPENDS? 

The  Best  Series  of  Manuals  for  the  Use  of  Students. 
Price  of  each,  Cloth,  .80.  Interleaved,  for  taking  Notes,  $1.25. 

These  Compends  are  based  on  the  most  popular  text-books 
and  the  lectures  of  prominent  professors,  and  are  kept  constantly  re- 
vised, so  that  they  may  thoroughly  represent  the  present  state  of  the 
subjects  upon  which  they  treat. 

The  authors  have  had  large  experience  as  Quiz-Masters  and 
attaches  of  colleges,  and  are  well  acquainted  with  the  wants  of  students. 

4®*  They  are  arranged  in  the  most  approved  form,  thorough  and 
concise,  containing  over  6oo  fine  illustrations,  inserted  wherever  they 
could  be  used  to  advantage. 

4^  Can  be  used  by  students  of  any  college. 

4^  They  contain  information  nowhere  else  collected  in  such  a 
condensed,  practical  shape.  Illustrated  Circular  free. 

No.  I.  POTTER.  HUMAN  ANATOMY.  Fifth  Revised  and 
Enlarged  Edition.  Including  Visceral  Anatomy.  Can  be  used 
with  either  Morris's  or  Gray's  Anatomy.  117  Illustrations  and  16 
Lithographic  Plates  of  Nerves  and  Arteries,  with  Explanatory 
Tables,  etc.  By  Samuel  O.  L.  Potter,  m.d..  Professor  of  the 
Practice  of  Medicine,  Cooper  Medical  College,  San  Francisco  ; late 
A.  A.  Surgeon,  U.  S.  Army. 

No.  2.  HUGHES.  PRACTICE  OF  MEDICINE.  Part  I.  Fifth 

Edition,  Enlarged  and  Improved,  By  Daniel  E.  Hughes, m.d., 
Physician-in-Chief,  Philadelphia  Hospital,  late  Demonstrator  of 
Clinical  Medicine,  Jefferson  Medical  College,  Phila. 

No.  3.  HUGHES.  PRACTICE  OF  MEDICINE.  Part  II. 

Fifth  Edition,  Revised  and  Improved.  Same  author  as  No.  2. 
No.  4.  BRUBAKER.  PHYSIOLOGY.  Eighth  Edition,  with 
new  Illustrations  and  a table  of  Physiological  Constants.  Enlarged 
and  Revised.  By  A.  P.  Brubaker,  m.d..  Professor  of  Physiology 
and  General  Pathology  in  the  Pennsylvania  College  of  Dental 
Surgery  ; Demonstrator  of  Physiology,  Jefferson  Medical  College, 
Philadelphia. 

No.  5.  LANDIS.  OBSTETRICS.  Fifth  Edition.  By  Henry  G. 
Landis,  m.d.  Revised  and  Edited  by  Wm.  H.  Wells,  m.d.. 
Assistant  Demonstrator  of  Obstetrics,  Jefferson  Medical  College, 
Philadelphia.  Enlarged.  47  Illustrations. 

No.  6.  POTTER.  MATERIA  MEDICA,  THERAPEUTICS, 
AND  PRESCRIPTION  WRITING.  Sixth  Revised  Edition 
(U,  S.  P.  1890).  By  Samuel  O.  L.  Potter,  m.d..  Professor  of 
Practice,  Cooper  Medical  College,  San  Francisco  ; late  A.  A.  Sur- 
geon, U.  S.  Army. 


MEDICAL  BOOKS. 


23 


7 QUIZ-COMPENDS  ?— Continued. 

No.  7.  WELLS.  GYNECOLOGY.  A New  Book.  By  Wm. 
H.  Wells,  M.D.,  Assistant  Demonstrator  of  Obstetrics,  Jefferson 
College,  Philadelphia.  Illustrated,  fust  Ready. 

No.  8.  GOULD  AND  PYLE.'  DISEASES  OF  THE  EYE 
AND  REFRACTION.  A New  Book.  Including  Treatment 
and  Surgery,  and  a Section  on  Local  Therapeutics.  By  George 
M.  Gould,  m.d.,  and  W.  L Pyle,  m.d.  With  Formulae,  Tables, 
and  III  Illustrations,  several  of  v^^hich  are  Colored. 

No.  9.  HORWITZ.  SURGERY,  Minor  Surgery,  and  Bandag- 
ing. Fifth  Edition,  Enlarged  and  Improved.  By  Orville 
Horwitz,  B.S.,  M.D.,  Clinical  Professor  of  Genito- Urinary  Surgery 
and  Venereal  Diseases  in  Jefferson  Medical  College  ; Surgeon  to 
Philadelphia  Hospital,  etc.  With  98  Formulae  and  71  Illustrations. 

No.  10.  LEFFMANN.  MEDICAL  CHEMISTRY.  Fourth 

Edition.  Including  Urinalysis,  Animal  Chemistry,  Chemistry  of 
Milk,  Blood,  Tissues,  the  Secretions,  etc.  By  Henry  Leffmann, 
M.D.,  Professor  of  Chemistry  in  Pennsylvania  College  of  Dental 
Surgery  and  in  the  Woman’s  Medical  College,  Philadelphia. 

No.  II.  STEWART.  PHARMACY.  Fifth  Edition.  Based  upon 
Prof.  Remington’s  Text-Book  of  Pharmacy.  By  F.  E.  Stewart, 
M.D.,  PH.G.,  late  Quiz-Master  in  Pharmacy  and  Chemistry,  Phila- 
delphia College  of  Pharmacy;  Lecturer  at  Jefferson  Medical 
College.  Carefully  revised  in  accordance  with  the  new  U.  S.  P. 

No.  12.  BALLOU.  VETERINARY  ANATOMY  AND  PHY- 
SIOLOGY. Illustrated.  By  Wm.  R.  Ballou,  m.d..  Professor 
of  Equine  Anatomy  at  New  York  College  of  Veterinary  Surgeons; 
Physician  to  Bellevue  Dispensary,  etc.  29  graphic  Illustrations. 

No.  13.  WARREN.  DENTAL  PATHOLOGY  AND  DEN- 
TAL MEDICINE.  Second  Edition,  Illustrated.  Containing 
a Section  on  Emergencies.  By  Geo.  W.  Warren,  d.d.s..  Chief 
of  Clinical  Staff,  Pennsylvania  College  of  Dental  Surgery. 

No.  14.  HATFIELD.  DISEASES  OF  CHILDREN.  Second 
Edition.  Colored  Plate.  By  Marcus  P.  Hatfield,  Profes- 
sor of  Diseases  of  Children,  Chicago  Medical  College. 

No.  15.  HALL.  GENERAL  PATHOLOGY  AND  MORBID 
ANATOMY.  91  Illustrations.  By  H.  Newberry  Hall,  ph.g., 
M.D.,  late  Professor  of  Pathology,  Chicago  Post-Graduate  Medi- 
cal School. 

Np.  16.  DISEASES  OF  THE  SKIN.  By  Jay  T.  Schamberg, 
M.D.,  Instructor  in  Skin  Diseases,  Philadelphia  Hospital.  Ulus. 

Price,  each,  Cloth,  .80.  Interleaved,  for  taking  Notes,  $1.25. 


In  preparing,  revising,  and  improving  Blakiston's  ? Quiz-Com- 
PENDS  ? the  particular  wants  of  the  student  have  always  been  kept  in 
mind. 

Careful  attention  has  been  given  to  the  construction  of  each  sentence, 
and  while  the  books  will  be  found  to  contain  an  immense  amount  of 
knowledge  in  small  space,  they  will  likewise  be  found  easy  reading ; 
there  is  no  stilted  repetition  of  words  ; the  style  is  clear,  lucid,  and  dis- 
tinct. The  arrangement  of  subjects  is  lystematic  and  thorough  ; there 
is  a reason  for  every  word.  They  contain  over  600  illustrations. 


Tvson’s 
Practice  of 
Medicine. 


A Text-Book  of  the  Practice  of  Medi- 
cine. With  Special  Reference  to  Diagnosis 
and  Treatment.  By  James  Tyson,  m.  d., 
Professor  of  Clinical  Medicine  in  the  Univer- 
sity of  Pennsylvania;  Physician  to  the  Hos- 
pital of  the  University  and  to  the  Philadelphia 
Hospital ; Fellow  of  the  College  of  Physicians 
of  Philadelphia,  etc. 


With  Many  Useful  Illustrations. 
Octavo.  ii8o  Pages. 

Cloth,  $5.50;  Sheep,  $6.50;  Half  Russia,  $7.50. 


Extracts  from  a Review  in  the  American  Journal  of 
Medical  Sciences ^ March,  1897: 

“ Externally  it  is  the  largest  and  handsomest  single  volume 
on  the  practice  of  medicine.” 

“ Clinical  features  are  usually  described  in  a masterly  way.” 
“The  directions  (for  treatment)  are  full  and  clear,  and  as 
a rule,  eminently  judicious  and  conservative.” 

“Dr.  Tyson's  style  is  already  so  well  known  in  medical 
literature  that  it  is  only  necessary  to  say  the  present  work  is 
one  of  the  best  examples.” 

“ We  welcome  Dr.  Tyson’s  Practice  as  a most  valuable 
addition  to  medical  literature.” 

Descriptive  circular  and  sample  pages  upon  application. 


